Rar selective agonists in combination with immune modulators for cancer immunotherapy

ABSTRACT

Disclosed herein are methods for treating cancer comprising administering CAR-modified immune cells and at least one Retinoic Acid Receptor agonist.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/117,372, filed Aug. 30, 2018, now U.S. Pat. No. ______, which claimsthe benefit of U.S. Provisional Patent Application No. 62/552,814, filedon Aug. 31, 2017, the entire contents of which are each incorporated byreference herein.

BACKGROUND

For years, the cornerstones of cancer treatment have been surgery,chemotherapy, and radiation therapy. Over the last decade, targetedtherapies—drugs that target cancer cells by homing in on specificmolecular changes seen primarily in those cells—have also emerged asstandard treatments for a number of cancers. A newer approach utilizingimmunotherapy involves engineering immune cells to recognize and attacktumors.

Normal hematopoietic stem cells (HSCs) are primed to be highly sensitiveto retinoids (compounds specific for the Retinoic Acid Receptor or RAR)but are maintained in a retinoid signaling-naïve state by isolating themfrom physiologic levels of retinoids. The bone marrow microenvironment,by expression of the enzyme CYP26 metabolically inactivates retinoicacid, regulates the exposure of the bone marrow to retinoids. Thismechanism (CPY26-mediated retinoid metabolism) is dynamic and used bythe bone marrow stroma to match HSC behavior to physiological needs. Forexample, steady state low levels of retinoids in the bone marrow nichemaintains HSCs in a quiescent state, while during situations of stress(i.e., exposure to radiation or chemotherapy) higher retinoid levels aremaintained to recruit HSCs into cell division and rescue hematopoiesis.

In subjects with hematologic malignancies, cancer HSCs are protectedfrom retinoids by stromal CYP26, in a similar fashion to the normalsituation. However, because of other alterations in the bone marrowniche in hematologic malignancies, such as differences in aldehydedehydrogenase (ALDH) activity, there exists a therapeutic window forretinoids to be useful in the treatment of hematologic malignancies.Expression of CYP26 by the bone marrow microenvironment contributes tothe protection of immature acute myeloid leukemia (AML) cells fromall-trans retinoic acid (ATRA) and may explain why ATRA is not effectivein treating AML. Exposure to pharmacological concentrations of ATRA,acting through retinoic acid receptor gamma (RARγ), induces CYP26expression in the bone marrow microenvironment, thus protecting thecancer stem cells therein from retinoid activity. This mechanism alsoshields non-hematopoietic metastatic tumor cells in the bone marrow.However, the use of retinoid analogs which are not inactivated by CYP26enables such retinoids to terminally differentiate, and thus eliminate,the cancer HSCs from the protective bone marrow niche. Since suchdifferentiation is mediated by RARα and the use of RARα specificanalogs, which are CYP26 resistant, enables the therapeuticdifferentiation-inducing activity without inactivation by the CYP26enzyme.

Thus, the combination of RAR agonists, to induce differentiation ofcancer stem cells, and targeted immunotherapy can be particularly usefulin treating cancer.

SUMMARY

Disclosed herein are compounds for potentiation of targeted cancerimmunotherapeutics. Compounds which act on retinoic acid receptors (RAR)are used in combination with chimeric antigen receptor (CAR)-modifiedimmune cells (sometimes abbreviated as CAR-MIC) to potentiate theanti-cancer activity of the CAR-modified immune cells and/or immunecheckpoint targeted therapeutics.

Retinoids and rexinoids have diverse activities. Particular retinoidshave an anticancer effect based in terminally differentiating cancerstem cells. These differentiating retinoids, or differentiating RARactive agents, can be used in combination with CAR-MIC to increase theoverall effectiveness of the immunotherapy. In some embodiments thedifferentiating retinoid is a RARα agonist. In some embodiments the RARαagonist is a RARα specific or selective agonist. In some embodiments theRARα agonists are CYP26 resistant.

In some embodiments, the CAR-modified immune cells are, or comprise,CAR-modified T cells. In some embodiments, the CAR-modified immune cellsare, or comprise, CAR-modified NK cells. In some embodiments, theCAR-modified immune cells are, or comprise, CAR-modified NKT cells. Insome embodiments, the CAR-modified immune cells are, or comprise,CAR-modified macrophages. Further embodiments can comprise mixtures ofthese cell types. Most typically such cellular preparations areadministered by infusion, for example intravenous infusion. In contrast,the RARα agonists are small molecules that can be administered orally,for example as pills or capsules and the like. Thus the RARα agonistsand the CAR-modified immune cells may be administered on independentschedules.

In some embodiments, the RARα agonist is:

In other embodiments, the RARα agonist is tamibarotene (AM80), AM580, orRe 80.

Other embodiments specifically exclude one or more of these RARαagonists.

In some embodiments, the cancer is a hematologic malignancy, such asacute myeloid leukemia or multiple myeloma. In some embodiments, thecancer is a solid tumor.

In some embodiments, the methods further comprise administering to thesubject at least one immune checkpoint inhibitor. In some embodiments,the immune checkpoint inhibitor is an inhibitor of at least one ofCTLA-4, PD-1, TIM-3, LAG-3, PD-L1 ligand, B7-H3, B7-H4, BTLA, or is anICOS or OX40 agonist. In some embodiments, the immune checkpointinhibitor is an antibody specific for at least one of CTLA-4, PD-1,TIM-3, LAG-3, PD-L1 ligand, B7-H3, B7-H4, BTLA, ICOS, or OX40. Otherembodiments specifically exclude one or more of these agents.

In some embodiments, the methods comprise additionally administering atleast one antineoplastic agent such as a cancer chemotherapy agent ortargeted therapy agent. In some embodiments the antineoplasitc agent isbortezomib.

Also disclosed herein are methods of prolonging the disease-freesurvival of a cancer patient comprising, administering CAR-modifiedimmune cells and at least one RARα agonist as compared to patientsreceiving CAR-modified immune cells but not receiving the RARα agonist.Other embodiments are methods of prolonging the progression-freesurvival or overall survival of a cancer patient comprisingadministering CAR-modified immune cells and at least one RARα agonist.

Also disclosed herein are methods of decreasing toxicity of CAR-modifiedimmune cells comprising administering to a subject in need thereof atleast one RARα agonist in combination with the CAR-modified immune cellssuch that as a result of the combination, a lower dose of CAR-modifiedimmune cells is administered more safely and equally effectively than ifthe CAR-modified immune cells were administered alone; or that a higherdose of CAR-MIC can be administered with greater efficacy and equalsafety.

Also disclosed herein are methods of treating cancer comprisingadministering to a subject in need thereof, chimeric antigen receptor(CAR)-modified immune cells, at least one RARα agonist, and at least oneimmune checkpoint inhibitor.

Also disclosed herein are methods of augmenting the production ofCAR-modified immune cells by inclusion of an RARα agonist in the culturemedium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the extent to which compound IRX5183 binds to andactivates transcription from RARα, RARβ, and RARγ using atransactivation assay.

FIG. 2A-C shows that RAR receptor specific agonists regulate FoxP3,α4β7, and CCR9 expression. Purified CD4⁺ CD25⁻ FoxP3⁻ cells werecultured in media with the specified concentration of each RAR agonistand analyzed by flow cytometry for FoxP3 (FIG. 2A), α4β7 (FIG. 2B), andCCR9 (FIG. 2C) expression in total CD4 T cells. FoxP3 results arerepresentative of three independent experiments. CCR9 and α4β7 resultsare representative of multiple experiments.

FIG. 3A-H depicts the relative concentration of plasma markers BCL6(FIGS. 3A and E), BLIMP-1 (FIGS. 3B and F), XBPS-1 (FIGS. 3C and G), andCHOP (FIGS. 3D and H) in multiple myeloma (MM) cell lines H929 (FIG.3A-D) or CD138+ MM cells from three different patient samples (FIG.3E-H) incubated for 5 days either in the absence of stroma (Liquid),with or without AGN (RA receptor antagonist AGN194310, 1 μM), orco-cultured with BM mesenchymal cells (Stroma), with or without R115(CYP26 inhibitor R115866, 1 μM) or IRX (CYP26-resistant retinoidIRX5183, 1 μM). Expression in untreated liquid conditions was set at 1.Data are representative of 3 independent experiments with similarresults and represent the mean±SEM. *P≤0.05 and **P≤0.01, byrepeated-measures 1-way ANOVA for determination of statisticalsignificance between groups; P values were corrected for multiplecomparisons using Dunnett's test. Ctrl, control; max, maximum.

FIG. 4A-B depicts the clonogenic recovery (CFU) of H929 cells (FIG. 4A)or cellular recovery of primary CD138+ MM cells from 3 different patientsamples (FIG. 4B). MM cells were treated with bortezomib (BTZ; 2.5 nM)for 48 hours after being incubated for 5 days either in the absence ofstroma (Liquid), with or without the pan-RAR inhibitor AGN (1 μM), or inthe presence of BM mesenchymal cells (Stroma), with or without the CYP26inhibitor R115 (1 μM) or the CYP26-resistant retinoid IRX (1 μM).Clonogenic or cellular recovery was normalized to each condition in theabsence of BTZ.

FIG. 5 depicts clonogenic recovery of H929 cells treated with BTZ (2.5nM). MM cells were incubated for 5 days in the absence (Liquid) orpresence of BM mesenchymal cells (Stroma), with or without R115 (1 μM).Following this preincubation, H929 cells were separated from BM stroma,cultured in fresh media for 0 to 48 hours, and then treated with BTZ(2.5 nM) for 48 hours. Clonogenic recovery was normalized to eachcondition in the absence of BTZ.

FIG. 6 depicts bioluminescent images of systemic MM xenografts.Following engraftment of H929 Luc+ cells, mice were treated with IRX(n=4), BTZ (n=5), or a combination of both (n=5) for 4 weeks. Datarepresent the mean±SEM of the fold change in bioluminescence(photons/second) from day 0.

FIG. 7A-C depicts the effects of MM cells on the expression of CYP26A1in BM stroma. Relative quantification of CYP26A1 mRNA in human BMmesenchymal cells incubated for 24 hours either in the absence (Ctrl) orpresence (Coculture or Transwell) of MM cells (H929 [FIG. 7A], MM.1S[FIG. 7B], U266 [FIG. 7C]). Expression in untreated BM stroma (Ctrl) wasarbitrarily set at 1.

FIG. 8A-C depicts the relative quantification of CYP26A1 mRNA in mousewild type (WT) or Smo-KO BM stroma incubated for 24 hr in the absence(Ctrl) or presence (Coculture or Transwell) of MM cells (H292, MM.1S,U266). Expression in untreated WT or Smo-KO stroma was arbitrarily setat 1 for the respective treated conditions. Data represent the mean±SEMof 3 independent experiments. *P≤0.05 and **P≤0.01, by unpaired,2-tailed Student's t test.

FIG. 9 depicts bioluminescent images of mice showing tumor burden during4 weeks of treatment with IRX (10 mg/kg), BTZ (0.5 mg/kg), or thecombination. Anterior tumors consisted of a combination of MM.1Sluciferase⁺ cells and Smo^(Fl/Fl) BM stroma cells transduced with acontrol vector (WT BM stroma). Posterior tumors consisted of acombination of MM.1S luciferase+ cells and Smo^(Fl/Fl) BM stroma cellstransduced with Cre-recombinase (Smo KO BM stroma).

FIG. 10 depicts the fold change in bioluminescence (photons/second) oftumors during 4 weeks of treatment. The change in bioluminescence foreach tumor at day 1 was normalized to the change in bioluminescence atday 14 and at the end of treatment (day 28).

FIG. 11A-D depicts the relative quantification of BCL6 (B cellmarker)(FIG. 11A), BLIMP (FIG. 11B), XBP1s (FIG. 11C), and CHOP (FIG.11D), (PC markers) in H929 cells from 3 different patient samplesincubated for 5 days either in the absence of stroma (Ctrl) orco-cultured with WT or Smo-KO stromal cells. Expression in untreatedliquid conditions was set at 1. Data represent the mean±SEM. *P≤0.05 and**P≤0.01, by repeated-measures 1-way ANOVA to determine statisticalsignificance between treatment groups; P values were corrected formultiple comparisons using Dunnett's test.

FIG. 12A-C depicts stroma blockage of ATRA-mediated, but not AM80- orIRX5183-induced, differentiation and elimination of AML. (FIG. 12A) CFUexperiments with NB4 cells treated with 10⁻⁷ M ATRA, IRX5183, or 10⁻⁸ MAM80; (FIG. 12B) OCI-AML3 cells and (FIG. 12C) Kasumi-1 cells treatedwith 10⁻⁶ M ATRA, IRX5183, or 10⁻⁷ M AM80 showed a decrease inclonogenic growth compared to control with AM80 and IRX5183 both off andon stroma. Data across three independent experiments.

DETAILED DESCRIPTION

Disclosed herein are combinations for therapy of cancer comprisingcoordinated administration of retinoid compounds and adoptive transferof immune cells expressing chimeric antigen receptors (CAR-modifiedimmune cells or CAR-MIC) and/or immune checkpoint targeted therapeutics.Compounds which act on retinoic acid receptors (RAR), in particular RARαagonists, can potentiate the activity of CAR-modified immune cells bycausing cancer stem cells to differentiate and leave the bone marrow ortumor site and become available to be attacked by the CAR-modifiedimmune cells.

By potentiation it is meant that the CAR-modified immune cells havegreater and/or more rapid effect when a RARα selective agonist is usedwith the CAR-modified immune cells than when a RARα agonist is not usedwith the CAR-modified immune cells or, similarly, that a given degree ofeffect can be obtained with a smaller dosage of CAR-modified immunecells when the RARα agonist is also used than would be required if theRARα selective agonist were not used.

As used herein, the term “potentiate” refers to an improved efficacy ofCAR-modified immune cells, or improved response by the patient, whenused in combination with a RARα agonist compared to the use ofCAR-modified immune cells in the absence of RARα agonist. As usedherein, the term “augment” also refers to an improved effect when usingan RARα agonist when compared to the situation where the RARα agonist isnot used.

Many, if not most, malignancies arise from a rare population of cellsthat exclusively maintain the ability to self-renew and sustain thetumor. These cancer stem cells are often biologically distinct from thebulk of differentiated cancer cells that characterize the disease. Forexample, chronic myeloid leukemia (CML) occurs at the level ofhematopoietic stem cells and, like their normal counterparts, CML stemcells undergo orderly differentiation. Thus, the bulk of the leukemicmass in CML consists of differentiated blood cells, whereas the rarecells responsible for disease maintenance resemble normal hematopoieticstem cells. Similarly, in multiple myeloma (MM), which is characterizedby neoplastic plasma cells, these cells appear to be terminallydifferentiated like their normal counterparts. The myeloma plasma cellsthat form the bulk of the tumor arise from a population of lessdifferentiated cancer stem cells that resemble post-germinal center Bcells. Other cancers, including but not limited to, hematologicalmalignancies, myelodysplastic syndrome, breast cancer, prostate cancer,pancreatic cancer, colon cancer, ovarian cancer, melanoma, non-melanomaskin cancers, and brain cancers have been demonstrated to arise fromcorresponding cancer stem cells.

Thus, disclosed herein are methods of treating cancer with agents whichcan target cancer stem cells in the protected bone marrow niche, orwithin tumors, by inducing differentiation of the cancer stem cells intomature cancer cells that are susceptible to therapy with CAR-modifiedimmune cells. Previous studies demonstrated that bone marrow stromalcells induce an immature drug-resistant phenotype in multiple myelomaand acute myeloid leukemia cells in the bone marrow. The bone marrowstroma creates a retinoic acid-low (RA-low) environment via CYP26 thatprevents the differentiation of normal and malignant cells. Sinceretinoid signaling promotes PC differentiation and Ig production,modulation of RA signaling is an attractive therapeutic strategy forovercoming drug resistance in the bone marrow microenvironment.Administration of RARα agonists which can act on the cancer stem cellsin the bone marrow niche (because they are not inactivated by CYP26),and cause differentiation of the cells (thus rendering them sensitive tokilling by CAR-modified immune cells) is one such approach. Suchdifferentiation can also be associated with a change in the cells frombeing resistant to sensitive to an anti-cancer drug. In some embodimentssuch an anti-cancer drug is bortezomib. In certain embodiments,effectiveness of therapy with a RARα agonist disclosed herein leads to asubstantial decrease in the number of cancer stem cells in the protectedenvironment.

In some embodiments disclosed herein are methods for treating cancerwith a combination of one or more RARα agonists and CAR-modified immunecells. Also disclosed herein are methods for treating cancer with acombination of one or more RARα agonists and one or more immunecheckpoint targeted cancer therapeutics. Also disclosed herein aremethods for treating cancer comprising one or more RARα agonists and oneor more immune checkpoint targeted cancer therapeutics and CAR-modifiedimmune cells.

RARα Agonists

Compounds with retinoid activity (vitamin A and its derivatives) haveactivity in cell proliferation and differentiation processes. Manybiological effects of retinoids are mediated by modulating the nuclearretinoic acid receptors (RARs). The RARs activate transcription bybinding to DNA sequence elements, known as RAR response elements (RARE),in the form of a heterodimer with one of the retinoid X receptors (knownas RXRs). Three subtypes of human RARs have been identified anddescribed: RARα, RARβ, and RARγ.

As used herein, the term “RARα selective agonist” refers to a compoundthat selectively binds RARα. As used herein, the term “selectivelybinds,” when made in reference to a RARα selective agonist, refers tothe discriminatory binding of a RARα selective agonist to the indicatedtarget RARα such that the RARα selective agonist does not substantiallybind with non-target receptors like a RARβ or a RARγ. While preferredembodiments make use of a RARα selective agonist, other embodiments canuse a RARα agonist that is not necessarily selective for RARα alone.Thus while many embodiments are described as using a RARα selectiveagonist it should be understood that otherwise similar embodiments usinga general RARα agonist are also disclosed.

The term “agonist” as used herein shall be understood to mean a compoundwhich binds to a receptor and activates it, producing gene transcriptionand a subsequent pharmacological response (e.g., contraction,relaxation, secretion, enzyme activation, etc.). As used herein, theterm “RARα agonist” refers to a compound that binds to RARα with asubstantially higher affinity compared to binding with another molecule,such as a different RAR. In exemplary embodiments, a RARα agonist isselective for RARα over RARγ and/or RARβ. A RAR selective agonist tendsto bind to a particular RAR receptor target with high binding affinityto the near effective exclusion of other RARs. As used herein, the term“agonist” includes selective agonists.

The term “antagonist” as used herein, refers to a compound thatattenuates the effect of an agonist by binding in the same site as anagonist without activating the receptor. An antagonist by itself willnot affect the gene transcriptional activity of the unoccupied receptor.Conventionally, a RARα antagonist is a chemical agent that inhibits theactivity of an RARα agonist. As used herein, the term “antagonist”includes selective antagonists.

The term “inverse agonist” as used herein shall be understood to mean acompound which produces an effect opposite to that of an agonist, yetacts at the same receptor. An inverse agonist by itself will reduce thebasal gene transcriptional activity of the unoccupied receptor.

Selective binding of a RARα agonist to a RARα includes bindingproperties such as, e.g., binding affinity and binding specificity.Binding affinity refers to the length of time a RARα agonist resides atits a RARα binding site, and can be viewed as the strength with which aRARα agonist binds RARα. Binding specificity is the ability of a RARαagonist to discriminate between a RARα and a receptor that does notcontain its binding site, such as, e.g., a RARβ or a RARγ. One way tomeasure binding specificity is to compare the association rate of a RARαagonist for its RARα relative to the association rate of a RARα agonistfor a receptor that does not contain its binding site; for example,comparing the association rate constant of a RARα agonist for its RARαrelative to a RARβ and/or a RARγ.

In some embodiments, a RARα agonist will have a ratio of activity at aRARα relative to a RARβ and/or a RARγ of, e.g., at least 5 timesgreater, at least 10 times greater, at least 15 times greater, at least20 times greater or at least 100 times greater. A RAR pan agonist willhave activity at a RARα, a RARβ, and a RARγ, i.e., similar affinity at aRARα, a RARβ, and a RARγ.

The binding specificity of a RARα agonist that selectively binds to aRARα can also be characterized as an activity ratio that such a RARαagonist can exert through binding to its RARα relative to a receptor notcomprising its binding site, such as, e.g., a RARβ or a RARγ. In someembodiments, a RARα agonist that selectively binds to a RARα has anactivity ratio through its RARα relative to a receptor not comprisingits binding site of, e.g., at least 2:1, at least 3:1, at least 4:1, atleast 5:1, at least 64:1, at least 7:1, at least 8:1, at least 9:1, atleast 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1,at least 35:1, or at least 40:1. In some embodiments, a RARα agonistthat selectively binds to a RARα has an activity ratio through its RARαrelative to a RARβ and/or a RARγ of, e.g., at least 2:1, at least 3:1,at least 4:1, at least 5:1, at least 64:1, at least 7:1, at least 8:1,at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least25:1, at least 30:1, at least 35:1, or at least 40:1.

In some embodiments, the RARα agonists useful in the methods disclosedherein are RARα agonists which are not metabolized by CYP26. CYP26 is acytochrome P450 monooxygenase that metabolizes retinoic acid intoinactive, or less active, substances which can also be readilyeliminated from cells and regulates cellular levels of retinoic acid.RARα selective agonists that are readily metabolized by CYP26 are notwithin the scope of these embodiments.

As used herein, the term “CYP26-resistant” refers to RARα agonists whichare not metabolized, degraded, or otherwise inactivated by the CYP26enzyme and have activity within the bone marrow.

In an aspect of this embodiment, a RARα agonist is a compound having thestructure of formula (I):

wherein R¹ is H or 01-6 alkyl, R² and R³ are independently H or F; and,R⁴ is a halogen.

In some embodiments of formula I, the halogen is F, Cl, Br or I. In someembodiments, of formula I, the halogen is F. In some embodiments, offormula I, the halogen is Cl. In some embodiments, of formula I, thehalogen is Br. In some embodiments, of formula I, the halogen is I.

In an aspect of this embodiment, a RARα agonist is a compound having astructure of formula (II):

wherein R¹ is H or C₁₋₆ alkyl.

In another aspect of this embodiment, a RARα agonist is the compoundhaving the structure of formula (III):

In any embodiment in which R¹ is C₁₋₆ alkyl, R¹ can be C₁ alkyl, C₂alkyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, C₆ alkyl, or any combinationthereof.

In another embodiment, the RARα agonist is tamibarotene (AM80;4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benzoicacid). In another embodiment, the RARα agonist is AM580(4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid). In another embodiment, the RARα agonist is Re 80(4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid).

Other RARα agonists useful as a compound disclosed herein are describedin U.S. Pat. Nos. 5,856,490; 5,965,606; and 6,387,950; each of which isincorporated by reference in its entirety. These references also presentdata to show that the compounds are indeed RARα agonists. Assays bywhich a compound can be tested and established to be a RARα agonist areknown in the art and are described in numerous prior art publicationsand patents. For example, a chimeric receptor transactivation assaywhich tests for agonist-like activity in the RARα, RARβ, RARγ, and RXRαreceptor subtypes, is described in detail in U.S. Pat. No. 5,455,265,which is hereby incorporated by reference in its entirety.

Aspects of the present specification provide, in part, a compositioncomprising a RARα agonist. A RARα agonist includes the compoundsdisclosed herein.

CAR-Modified Immune Cells

Tumor cells often down-regulate major histocompatibility complex (MHC)expression and furthermore, when they do express MHC alleles, theimmunodominant epitopes are not often known. Thus, MHC-dependent cancerimmunotherapies are often not effective. Chimeric antigen receptor(CAR)-modified immune cells react with target antigens on cancer cellsin an MHC-independent matter. The CAR allows binding via theantigen-binding domain to target cells wherein the CAR-modified cellskill the target cells in a MHC non-restricted manner by binding to thetarget cells and induction of activation and cytotoxicity of themodified cells against the tumor target. Binding to target cells canalso induce proliferation of the CAR-modified cells.

As used herein, the term “target cells” refers to cells expressing asurface antigen that can be bound by the CAR. The antigen can also bereferred to as the “target antigen.” Target antigens are antigens thatare differentially expressed on cancer cells such that the CAR targetsthe cancer cells preferentially over non-cancer cells.

Once the modified immune cells bind to target antigen, the internalstimulatory domains of the CAR provide the necessary signals for theimmune cell to become fully active. In this fully active state, theimmune cells can more effectively proliferate and attack cancer cells.

CAR-modified cells can recognize a variety of types of antigen, not onlyprotein but also carbohydrate and glycolipid structures typicallyexpressed on the tumor cell surface. Unlike T cell receptor (TCR)recognition, the antigen does not need to be processed and presented byMHC and therefore the same CAR-molecule can be used in all patients whoexpress the same tumor antigen regardless of HLA type.

The CAR comprises a recombinant polypeptide construct comprising atleast an antigen-binding domain, a transmembrane domain, and one or moreintracellular stimulatory domains (also referred to as a cytoplasmicsignaling domain or an intracellular signaling domain). Theantigen-binding domain allows the modified immune cells to specificallybind to the target antigen, the transmembrane domain anchors the CAR inthe plasma membrane of the immune cells, and the intracellularstimulatory domain induces persistence, trafficking, and effectorfunctions in the transduced cells.

The antigen-binding domain of a CAR is often derived from a monoclonalantibody, but other ligands (e.g., heregulin, cytokines) and receptors(e.g., NKp30) can also be used. The antigen-binding domain can includean antibody, or a fragment of an antibody that retains antigen-bindingfunction. For example, the CAR antigen-binding domain is oftencontributed by a single-chain variable fragment (scFv), which is formedfrom the variable regions of heavy and light chains of an antibody.

In one aspect, the transmembrane domain comprises a sequence of the zeta(ζ) chain associated with the T cell receptor complex, such as theintracellular domain of human CD3 ζ chain.

The one or more intracellular stimulatory domains of the CAR can includean intracellular stimulatory domain of one or more of CD28, 4-1BB(CD137), CD134 (OX-40), ICOS, and CD40L.

The antigen-binding domain, transmembrane domain, and the intracellularstimulatory domain(s) are linked either directly or via a spacersequence.

The CAR sequences are incorporated in an expression vector. Variousexpression vectors are known in the art and any such vector may beutilized. In some embodiments, the vector will be a retroviral orlentiviral vector. In other embodiments the vector will be derived fromadeno-associated virus.

Immune cells are transformed with the CAR and the CAR is then expressedon the cell surface. Typically, the immune cell stably expresses theCAR, although in some embodiments, the immune cell may transientlyexpress the CAR. The immune cell is thus transfected with a nucleicacid, e.g., mRNA, cDNA, DNA, encoding a CAR. Immune cells of thedisclosure include mammalian cells (e.g., human cells), and can beautologous cells, syngeneic cells, allogenic cells, and even in somecases, xenogeneic cells, The cells are engineered to express a CAR and,therefore like the CAR itself, are not found in nature. Exemplary immunecells include T lymphocytes (T cells), natural killer (NK) cells, NKTcells, and macrophages (including monocytes and dendritic cells).

The CAR-modified immune cells are then cultured to expand the populationand obtain a suitable number of cells for a single dose or for multipledoses.

In certain embodiments, one or more retinoid and/or rexinoid activeagents (e.g. a RARα antagonist, a RARγ agonist, a RXR antagonist, orcombinations thereof) are added to the expansion cultures during theculture period and have an effect on the CAR-modified cells directly.For example, in culturing CAR-MIC the one or more retinoid and/orrexinoid active agents added to the expansion cultures would be chosenfor their ability to, for example, suppress the development of Tregcells and/or their ability to promote the development Th17 cells. Insome embodiments, the one or more retinoid and/or rexinoid active agentsare included in the expansion culture of CAR-modified immune cells andadministered directly to a subject. This use of retinoid and/or rexinoidactive agents is described in U.S. patent application Ser. Nos.16/034,064 and 16/034,123 which are incorporated by reference herein forall that they teach related to this use.

In certain embodiments, one or more RARα agonists are added to theexpansion cultures during the culture period and have an effect on theCAR-modified cells directly. In some embodiments, the one or more RARαagonists are included in the expansion culture of CAR-modified immunecells and administered directly to a subject.

Immune Checkpoint Targeted Cancer Therapeutics

Immune checkpoint therapy targets regulatory pathways in thedifferentiation and activation of T cells to promote the passage of Tcell developmental program through these checkpoints so that anti-tumor(or other therapeutic) activity can be realized. The agents bringingabout immune checkpoint therapy are commonly called immune checkpointinhibitors and it should be understood that it is the check on T celldevelopment that is being inhibited. Thus, while many immune checkpointinhibitors also inhibit the interaction of receptor-ligand pairs (e.g.,anti-PD-1, anti-PD-L1, and CTLA-4), others (such as anti-OX40 andanti-ICOS) act as agonists of targets that release or otherwise inhibitthe check on T cell development, ultimately promoting effector functionand/or inhibiting regulatory function.

Disclosed herein is the use of immune checkpoint inhibitor molecules incombination with CAR-modified immune cells and RARα agonists. Moleculeswhich inhibit immune checkpoint proteins include antibodies which arespecific to one or more of PD-1, PD-1 ligand, CTLA-4, TIM-3, LAG-3,B7-H3, and B7-H4.

Programed death-1 (PD-1) is a checkpoint protein on T cells and normallyacts as a type of “off switch” that helps keep the T cells fromattacking other cells in the body. It does this by binding to programmeddeath ligand-1 (PD-L1), a protein on some normal and cancer cells. WhenPD-1 binds to PD-L1, the T cells will not attack the target cells. Somecancer cells have large amounts of PD-L1, which helps them evade immuneattack. Monoclonal antibodies (mAbs) that target either PD-1 or PD-L1can boost the immune response against cancer cells and have shown agreat deal of promise in treating certain cancers. Examples ofmonoclonal antibodies that target PD-1/PD-L1 include: the anti-PD-1 mAbsnivolumab (OPDIVO®, Bristol-Myers Squibb) and pembrolizumab (KEYTRUDA®,Merck & Co.), BMS-936559 (Bristol-Myers Squibb), pidilizumab(Medivation), and the anti-PD-L1 mAbs durvalumab (MED14736, IMFINZI™,Medimmune), atezolizumab (MPDL3280A; TECENTRIQ®, Hoffman-La Roche), andavelumab (BAVENCIO®, EMD Serono). These antibodies have, variously,demonstrated utility in treating a variety of cancers includingmalignant melanoma (MM), renal cell carcinoma (RCC), Merkel cellcarcinoma, urothelial carcinoma, and non-small cell lung cancer (NSCLC).Non-antibody inhibitors of PD-1/PD-11 interaction are also beingdeveloped; for example, small engineered proteins based on stefin A(called AFFIMER® molecules). In addition to PD-L1, PD-1 can also bind toPD-L2. In addition to PD-1, PD-L1 can also bind to B7-1 (CD80).

CTLA-4 is an immune checkpoint molecule expressed on the surface of CD4and CD8 T cells and on CD25+FOXP3+ T regulatory (Treg) cells. CTLA-4generates inhibitory signals that block T cell responses and enablestumor growth. Anti-CTLA-4 mAbs such as ipilimumab (YERVOY®;Bristol-Myers Squibb) cause shrinkage of tumors in animal models.Ipilimumab improves overall survival in MM patients and is approved forthe treatment of MM. Responses have been observed in renal cell cancer(RCC) and non small cell lung cacner (NSCLC) as well. Other exemplaryanti-CTLA-4 antibodies include tremelimumab (Medimmune).

TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) is amolecule selectively expressed on IFN-γ-producing CD4⁺ T helper 1 (Th1)and CD8⁺ T cytotoxic 1 (Tc1) T cells. TIM-3 is an immune checkpointreceptor that functions specifically to limit the duration and magnitudeof Th1 and Tc1 T-cell responses. Exemplary antibodies to TIM-3 aredisclosed in U.S. Patent Application Publication 20160075783 which isincorporated by reference herein for all it contains regardinganti-TIM-3 antibodies.

LAG-3 (lymphocyte-activation gene 3; CD223) negatively regulatescellular proliferation, activation, and homeostasis of T cells, in asimilar fashion to CTLA-4 and PD-1 and plays a role in Treg suppressivefunction. Exemplary antibodies to LAG-3 include GSK2831781(GlaxoSmithKline), BMS-986016 (Bristol-Myers Squibb,) and the antibodiesdisclosed in U.S. Patent Application Publication 2011/0150892 which isincorporated by reference herein for all it contains regardinganti-LAG-3 antibodies.

The B7 family of costimulatory proteins are expressed on the surface ofantigen-presenting cells and interact with ligands on T cells. B7-H3(CD276) is one of the molecules in this family. An antibody to B7-H3,enoblituzumab (EMPLICITI™, Bristol-Myers Squibb) is approved fortreatment of multiple myeloma. Another molecule in the family is B7-H4(V-set domain-containing T-cell activation inhibitor 1), antibodiesagainst which are in development.

Other immune checkpoint inhibitor targets, B- and T-cell attenuator(BTLA), inducible T-cell costimulator (ICOS), OX40 (tumor necrosisfactor receptor superfamily, member 4), and others, are potentiallyuseful in the disclosed methods. Several anti-OX40 agonistic mAbs are inearly phase cancer clinical trials including MED10562 and MED16469(Medimmune), MOXR0916 (Genetech), and PF-04518600 (Pfizer); as is ananti-ICOS agonistic antibody, JTX-2011 (Jounce Therapeutics).

Disclosed herein are methods of treating cancer comprising administeringCAR-modified immune cells, one or more RARα agonists, and one or moreimmune checkpoint targeting immunotherapeutics including a CTLA-4inhibitor, a PD-1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, aPD-1 ligand (such as PD-L1), an inhibitor of a PD-1 ligand, an OX40agonist, an ICOS agonist, a B7-H3 protein, an inhibitor of a B7-H3protein, a B7-H4 protein, and an inhibitor of a B7-H4 protein. Incertain embodiments, the immune checkpoint inhibitors are antibodies.

The immune checkpoint targeting immunotherapeutic antibodies can bewhole antibodies or antibody fragments. The terms “fragment of anantibody,” “antibody fragment,” and “functional fragment of an antibody”are used interchangeably herein to mean one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Theantibody fragment desirably comprises, for example, one or morecomplementary determining regions (CDRs), the variable region (orportions thereof), the constant region (or portions thereof), orcombinations thereof. Examples of antibody fragments include, but arenot limited to, a Fab fragment, which is a monovalent fragmentconsisting of the V_(L), V_(H), C_(L), and CH₁ domains; a F(ab′)₂fragment, which is a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a single chain Fv, in which the V_(L) and V_(H) domains arejoined by a peptide linker sequence; a Fab′ fragment, which results frombreaking the disulfide bridge of an F(ab′)₂ fragment using mild reducingconditions; a disulfide-stabilized Fv fragment (dsFv); and a domainantibody (dAb), which is an antibody single variable region domain (VHor VL) polypeptide that specifically binds antigen. It should also berealized that any of these forms of antigen-binding antibody fragmentscan provide the antigen binding domain of a CAR.

In alternative embodiments, the immune checkpoint inhibitor antibody isreplaced with another protein that similarly binds to the immunecheckpoint target molecule. In some instances these non-antibodymolecules comprise an extracellular portion of the immune checkpointtarget molecule's ligand or binding partner, that is, at least theextracellular portion needed to mediate binding to the immune checkpointtarget molecule. In some embodiments this extracellular binding portionof the ligand is joined to additional polypeptide in a fusion protein.In some embodiments the additional polypeptide comprises an Fc orconstant region of an antibody.

Methods of Treatment

Provided herein are methods of treating cancer in a mammal byadministering one or more RARα agonists and CAR-modified immune cells.In some embodiments, immune checkpoint inhibitors are administered inaddition to the CAR-modified immune cells and one or more RARα agonists.Also provided are methods of decreasing tumor burden and/or increasingthe disease-free or progression-free survival in subject with cancer.Other embodiments relate to compositions comprising such agents for usein the treatment of cancer and for use in making medicaments for thetreatment of cancer. It is to be understood that the multiple agentsused may be provided in separate compositions or medicaments which maybe administered by separate routes of administration and/or at separatetimes; nonetheless use of such multiple compositions or medicaments iscoordinated so that the patient to whom they are administered receivesthe benefit of the combined, interacting activity of the multipleagents. For each method of treating cancer disclosed herein there arecorresponding methods of cancer immunotherapy. For each method oftreating cancer or cancer immunotherapy there are corresponding methodsof potentiating cancer treatment/immunotherapy.

In various embodiments one or more RARα agonists are administered to asubject receiving or scheduled to receive CAR-modified immune cells oran immune checkpoint inhibitor. In these embodiments thesedifferentiating RARα agonists are administered prior to and/or duringthe interval in which the CAR-modified immune cells or an immunecheckpoint inhibitor is present in the subject. It is preferred that theRARα agonist is CYP26-resistant.

In some embodiments, the method comprises administering one or more RARαagonists and CAR-modified immune cells. In some embodiments, the methodcomprises administering one or more RARα agonists and one or more immunecheckpoint inhibitors. In yet other embodiments, the method comprisesadministering one or more RARα agonist, CAR-modified immune cells, andone or more RARα agonists and one or more immune checkpoint inhibitors.In certain embodiments, the RARα agonist is IRX5183 (AGN195183). Withrespect to the use of multiple RARα agonists in the various use ormethod of treatment embodiments described herein, any of the disclosedgeneral formula genera, sub-genera thereof, and individual species maybe combined with any other general formula genera, sub-genera thereof,and individual species, each such combination defining an individualembodiment.

The compounds, pharmaceutical compositions, and methods disclosed hereinare particularly useful for the treatment of cancer. As used herein, theterm “cancer” refers to a cellular disorder characterized byuncontrolled or dysregulated cell proliferation, decreased cellulardifferentiation, inappropriate ability to invade surrounding tissue,and/or ability to establish new growth at ectopic sites. The term“cancer” includes, but is not limited to, solid tumors and hematologictumors. The term “cancer” encompasses diseases of skin, tissues, organs,bone, cartilage, blood, and vessels. The term “cancer” furtherencompasses primary and metastatic cancers. Included within the term“cancer cells” are cancer stem cells.

The disclosed methods can be used to treat any type of cancer known inthe art. In certain embodiments, the cancer is a hematologic malignancy.Non-limiting examples of hematologic malignancy include acute myeloidleukemia, chronic myelogenous leukemia (CML), including accelerated CMLand CML blast phase, acute lymphoblastic leukemia, chronic lymphocyticleukemia, Hodgkin's disease, non-Hodgkin's lymphoma, includingfollicular lymphoma and mantle cell lymphoma, B-cell lymphoma, T-celllymphoma, multiple myeloma, Waldenstrom's macroglobulinemia,myelodysplastic syndromes, including refractory anemia, refractoryanemia with ringed sideroblasts, refractory anemia with excess blasts(RAEB), and RAEB in transformation, and myeloproliferative syndromes.

In some embodiments, the cancer is a solid tumor. In other embodiments,the cancer is a solid tumor which can metastasize to the bone.Non-limiting examples of solid tumors that can be treated by thedisclosed methods include pancreatic cancer, bladder cancer, colorectalcancer, breast cancer, prostate cancer (e.g., androgen-dependent andandrogen-independent prostate cancer), renal cancer, hepatocellularcancer, lung cancer (e.g., non-small cell lung cancer (NSCLC), smallcell lung cancer (SCLC), bronchoalveolar carcinoma (BAC), andadenocarcinoma of the lung), ovarian cancer (e.g., progressiveepithelial or primary peritoneal cancer), cervical cancer, gastriccancer, esophageal cancer, head and neck cancer (e.g., squamous cellcarcinoma of the head and neck), melanoma, neuroendocrine cancer, braintumors (e.g., glioma, anaplastic oligodendroglioma, adult glioblastomamultiforme, and adult anaplastic astrocytoma), bone cancer, and softtissue sarcoma.

In select embodiments a particular type of cancer is treated. In otherselect embodiments a particular type of cancer is excluded fromtreatment.

Additionally, the one or more RARα agonists can decrease toxicityassociated with CAR-modified immune cells by allowing a lower dose ofCAR-modified immune cells to be administered with the same efficacy or ahigher dose of the CAR-modified immune cells can be administered withthe same degree of safety. Intermediate doses between the lower, sameefficacy dose and the higher, same safety dose are also envisioned. Thususe of a RARα agonist in coordination with CAR-modified immune cellcancer immunotherapy can lead to the reduction or avoidance of thefrequency or severity of toxicities and adverse events related to theactivity of the CAR-modified immune cells, or at least the absence of aclinically-relevant increase. By improved or same safety it is meantthat the frequency, severity, or both, of one or more toxic or adverseevents related to use of CAR-modified immune cells is reduced or atleast not increased, respectively. It is to be understood that in thetreatment of life-threatening diseases, such as cancer, treatments withpotential for substantial toxicity can be considered sufficiently safe.

The cancer stem cells can be enumerated by various mechanisms andreduction in their numbers as a result of administration of aCYP26-resistant RARα agonist measured thereby. In embodiments disclosedherein, as a result of administration of a RARα agonist, the cancer stemcells in the bone marrow are reduced by more than about 0.5 log, morethan about 1 log, more than about 1.5 log, more than about 2.0 log, morethan about 2.5 log, more than about 3.0 log, more than about 3.5 log,more than about 4.0 log, more than about 4.5 log, or more than about 5.0log.

The term “treating” or “treatment” broadly includes any kind oftreatment activity, including the diagnosis, mitigation, or preventionof disease in man or other animals, or any activity that otherwiseaffects the structure or any function of the body of man or otheranimals. Treatment activity includes the administration of themedicaments, dosage forms, and pharmaceutical compositions describedherein to a patient, especially according to the various methods oftreatment disclosed herein, whether by a healthcare professional, thepatient him/herself, or any other person. Treatment activities includethe orders, instructions, and advice of healthcare professionals such asphysicians, physician's assistants, nurse practitioners, and the like,that are then acted upon by any other person including other healthcareprofessionals or the patient him/herself. In some embodiments, treatmentactivity can also include encouraging, inducing, or mandating that aparticular medicament, or combination thereof, be chosen for treatmentof a condition—and the medicament is actually used—by approvinginsurance coverage for the medicament, denying coverage for analternative medicament, including the medicament on, or excluding analternative medicament, from a drug formulary, or offering a financialincentive to use the medicament, as might be done by an insurancecompany or a pharmacy benefits management company, and the like. In someembodiments, treatment activity can also include encouraging, inducing,or mandating that a particular medicament be chosen for treatment of acondition—and the medicament is actually used—by a policy or practicestandard as might be established by a hospital, clinic, healthmaintenance organization, medical practice or physicians group, and thelike.

A typical dose of CAR-modified immune cells can be, for example, in therange of 1×10⁶ to 3×10¹⁰ cells per dose. In some embodiments,CAR-modified immune cells are administered at a dose of at least 1×10⁶cells/dose, at least 3×10⁶ cells/dose, at least 1×10⁷ cells/dose, atleast 3×10⁷ cells/dose, at least 1×10⁸ cells/dose, at least 3×10⁸cells/dose, at least 1×10⁹ cells/dose, at least 3×10⁹ cells/dose, atleast 1×10¹⁰ cells/dose, at least 3×10¹⁰ cells/dose, or a range definedby any two of the foregoing values. In some embodiments, the typicaldose of CAR-modified immune cells can be, for example, in the range of1×10⁵ to 1×10⁸ cells per kilogram of patient body weight. In someembodiments, CAR-modified immune cells are administered at a dose of atleast 1×10⁵ cells/kg, at least 3×10⁵ cells/kg, at least 6×10⁵ cells/kg,at least 1×10⁶ cells/kg, at least 3×10⁶ cells/kg, at least 6×10⁶cells/kg, at least 1×10⁷ cells/kg, at least 3×10⁷ cells/kg, or a rangedefined by any two of the foregoing values.

Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment can berepeated until a desired suppression of disease or disease symptomsoccurs. However, other dosage regimens may be useful and are within thescope of the present disclosure. The desired dosage can be delivered bya single bolus administration, by multiple bolus administrations, or bycontinuous infusion administration of the CAR-modified immune cells. Invarious embodiments the continuous infusion may extend for half an hour,for an hour, for several hours, for a day, or for several days.Treatment may comprise a single or multiple infusions.

In some embodiments, the CAR-modified immune cells are administered withother pre-treatment or simultaneous administrations of additionalagents. In some embodiments, subjects who are to receive CAR-modifiedimmune cells are pre-treated with a nonmyeloablative,lymphocyte-depleting regiment, such as, but not limited to, treatmentwith cyclophosphamide and/or fludarabine. In some embodiments,CAR-modified immune cells are administered with interleukin-2.

CAR-modified immune cells may be administered to a subject a single timeor multiple times. The cells can be administered weekly, biweekly,monthly, bimonthly, or upon evidence of cancer progression.

“Administering”, as used herein, refers to providing a pharmaceuticalagent or composition to a subject, and includes, but is not limited to,administering by a medical professional and self-administering.Administration includes, but is not limited to, oral administration,nasal administration, pulmonary administration, subcutaneousadministration, intravenous administration, intramuscularadministration, intratumoral administration, intracavity administration,intravitreal administration, dermal administration, and transdermaladministration, etc.

Depending on the type of cancer, and the patient to be treated, as wellas the route of administration, the disclosed RARα selective agonistsmay be administered at varying therapeutically effective doses to apatient in need thereof.

However, the dose administered to a mammal, particularly a human, in thecontext of the present methods, should be sufficient to effect atherapeutic response in the mammal over a reasonable timeframe. Oneskilled in the art will recognize that the selection of the exact doseand composition and the most appropriate delivery regimen will also beinfluenced by inter alia the pharmacological properties of theformulation, the nature and severity of the condition being treated, andthe physical condition and mental acuity of the recipient, as well asthe potency of the specific compound, the age, condition, body weight,sex and response of the patient to be treated, and the stage/severity ofthe disease.

As a non-limiting example, when administering a RARα agonist disclosedherein to a mammal, a therapeutically effective amount generally may bein the range of about 1 mg/m²/day to about 100 mg/m²/day. In someembodiments, an effective amount of a RARα agonist disclosed herein maybe about 5 mg/m²/day to about 90 mg/m²/day, about 10 mg/m²/day to about80 mg/m²/day, about 15 mg/m²/day to about 70 mg/m²/day, about 20mg/m²/day to about 65 mg/m²/day, about 25 mg/m²/day to about 60mg/m²/day, or about 30 mg/m²/day to about 55 mg/m²/day. In someembodiments, a therapeutically effective amount of a compound or acomposition disclosed herein may be at least 10 mg/m²/day, at least 15mg/m²/day, at least 20 mg/m²/day, at least 25 mg/m²/day, at least 30mg/m²/day, at least 35 mg/m²/day, at least 40 mg/m²/day, at least 45mg/m²/day, at least 50 mg/m²/day, at least 55 mg/m²/day, at least 60mg/m²/day, at least 65 mg/m²/day, or at least 75 mg/m²/day. In someembodiments, a therapeutically effective amount of a RARα agonistdisclosed herein may be at most 15 mg/m²/day, at most 20 mg/m²/day, atmost 25 mg/m²/day, at most 30 mg/m²/day, at most 35 mg/m²/day, at most40 mg/m²/day, at most 45 mg/m²/day, at most 50 mg/m²/day, at most 55mg/m²/day, at most 60 mg/m²/day, at most 65 mg/m²/day, at most 70mg/m²/day, at most 80 mg/m²/day, at most 90 mg/m²/day, or at most 100mg/m²/day.

The average surface area of a human body is generally accepted to be 1.9m² for an adult male, 1.6 m² for an adult female, and 1.33 m² for a12-13 year old child. These values can be used to calculate dose rangesfor daily dosage for the values in the preceding paragraph. The totaldaily dosage of RARα agonist can be administered as a single dose or astwo doses administered with a 24 hour period spaced 8 to 16, or 10 to14, hours apart. The RARα agonist(s) are administered in coordinationwith the CAR-modified immune cells and as above therapeutic orprophylactic efficacy can be monitored by periodic assessment of treatedpatients. For repeated administrations over several days or longer,depending on the condition, the treatment can be repeated until adesired suppression of disease or disease symptoms occurs. However,other dosage regimens may be useful and are within the scope of thedisclosure. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

Administration may be continuous or intermittent. The dosage may also bedetermined by the timing and frequency of administration. Thus, the RARαagonists disclosed herein can be given on a daily, weekly, biweekly, ormonthly basis for a period of time, followed by an optional drug holiday(drug free period) and that this drug administration/drug holiday cyclecan be repeated as necessary. In certain embodiments, the total dailydosage of RARα agonists can be administered as a single dose or as twodoses administered with a 24 hour period spaced 8 to 16, or 10 to 14,hours apart.

The RARα agonists are administered in coordination with the CAR-modifiedimmune cells and as above therapeutic or prophylactic efficacy can bemonitored by periodic assessment of treated patients. For repeatedadministrations over several days or longer, depending on the condition,the treatment can be repeated until a desired suppression of disease ordisease symptoms occurs. However, other dosage regimens may be usefuland are within the scope of the disclosure. The desired dosage can bedelivered by a single bolus administration of the composition, bymultiple bolus administrations of the composition, or by continuousinfusion administration of the composition.

The RARα agonists can be administered to a mammal using standardadministration techniques, including parenteral, oral, intravenous,intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. The term“parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. TheCAR-modified immune cells are administered to a mammal by intravenous,intraperitoneal, or subcutaneous injection. The RARα agonist preferablyis suitable for oral administration, for example as a pill, tablet orcapsule.

In one embodiment, the RARα agonist is administered daily for a periodof time, i.e., at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, at least 1 week, at least 2 weeks, atleast 3 weeks, or at least one month, before administration ofCAR-modified immune cells. In some embodiments, the RARα agonist isadministered starting within 1-7 days of obtaining the patient'speripheral blood lymphocytes for generation of autologous CAR-modifiedimmune cells. CAR-modified immune cells will generally persist in thebody for extended periods of time, much longer than will a RARα agonist.It is anticipated that RARα agonist therapy will be administered on adaily basis for a period of time and may be given longer thanCAR-modified immune cells. In some embodiments, the RARα is administeredfor longer than the CAR-modified immune cells, such as for a total of 30days, 90 days, 1 year, 2 years, 5 years, or more. Alternatively,administration of the RARα agonist may not continue throughout the wholeperiod in which the CAR-modified immune cells persist in the body, andmay cease up to a week before, within a day before or after, at the timeof, or any of 1-14 days after, administration of the CAR-modified immunecells. The treatment may be conducted in multiple cycles, in which caseadministration of the RARα agonist can resume prior to the expectedadministration of CAR-modified immune cells as described above.

Furthermore, CAR-modified immune cells are administered as a singlebolus administration, or with daily, weekly, or monthly administrationsof 0.5-20×10⁶ cells per administration.

The CAR-modified immune cells and RARα agonists disclosed herein may beadministered in combination with other drugs, such as at least one otheranticancer/antineoplastic agent including, for example, anychemotherapeutic agent or targeted therapy agent known in the art,ionizing radiation, small molecule anticancer agents, cancer vaccines,biological therapies (e.g., other monoclonal antibodies, cancer-killingviruses, gene therapy, and adoptive T-cell transfer), and/or surgery. Inother embodiments the CAR-modified immune cells and RARα agonists arethe only therapeutic reagents administered or the only treatment given;or the only treatment or reagents given, the primary utility of which isto promote an anti-cancer immune response.

In some exemplary embodiments treatment is initiated with administrationof a RARα agonist. In some embodiments the RARα agonist isCYP26-resistant. Concurrent with or subsequent to administration of theRARα agonist, CAR-MIC, and optionally an antineoplastic agent, isadministered, concurrently or sequentially. In some aspects of theseembodiments the antineoplastic agent, if used, is administered prior toadministration of the CAR-MIC. The antineoplastic agent is administeredfor an interval of 1 day to 1 month, for example, 2 days, 5 days, 1, 2,3, or 4 weeks. In some embodiments, administration of the antineoplasticagent ceases 1 day to 1 week prior to administration of the CAR-MIC. Insome embodiments the CAR-MIC is a CAR-T cell. In some embodiments theantineoplastic agent is bortezomib. In various aspects of theseembodiments the cancer can be a myeloma, such as multiple myeloma, or aleukemia, such as AML, CML, or acute promyelocytic leukemia (APL). In afurther aspect of these embodiments the RARα agonist can be formula(III) (IRX5183).

The effectiveness of cancer therapy is typically measured in terms of“response.” The techniques to monitor responses can be similar to thetests used to diagnose cancer such as, but not limited to:

-   -   A lump or tumor involving some lymph nodes can be felt and        measured externally by physical examination.    -   Some internal cancer tumors will show up on an x-ray or CT scan        and can be measured with a ruler.    -   Blood tests, including those that measure organ function can be        performed.    -   A tumor marker test can be done for certain cancers.

Regardless of the test used, whether blood test, cell count, or tumormarker test, it is repeated at specific intervals so that the resultscan be compared to earlier tests of the same type.

Response to cancer treatment is defined several ways:

-   -   Complete response—all of the cancer or tumor disappears; there        is no evidence of disease. Expression level of tumor marker (if        applicable) may fall within the normal range.    -   Partial response—the cancer has shrunk by a percentage but        disease remains. Levels of a tumor marker (if applicable) may        have fallen (or increased, based on the tumor marker, as an        indication of decreased tumor burden) but evidence of disease        remains.    -   Stable disease—the cancer has neither grown nor shrunk; the        amount of disease has not changed. A tumor marker (if        applicable) has not changed significantly.    -   Disease progression—the cancer has grown; there is more disease        now than before treatment. A tumor marker test (if applicable)        shows that a tumor marker has risen.

Other measures of the efficacy of cancer treatment include intervals ofoverall survival (that is time to death from any cause, measured fromdiagnosis or from initiation of the treatment being evaluated)),cancer-free survival (that is, the length of time after a completeresponse cancer remains undetectable), and progression-free survival(that is, the length of time after disease stabilization or partialresponse that resumed tumor growth is not detectable).

There are two standard methods for the evaluation of solid cancertreatment response with regard to tumor size (tumor burden), the WHO andRECIST standards. These methods measure a solid tumor to compare acurrent tumor with past measurements or to compare changes with futuremeasurements and to make changes in a treatment regimen. In the WHOmethod, the solid tumor's long and short axes are measured with theproduct of these two measurements is then calculated; if there aremultiple solid tumors, the sum of all the products is calculated. In theRECIST method, only the long axis is measured. If there are multiplesolid tumors, the sum of all the long axes measurements is calculated.However, with lymph nodes, the short axis is measured instead of thelong axis.

In some embodiments of the current method, the tumor burden of a treatedpatient is reduced by about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about95%, about 100%, or any range bound by these values.

In other embodiments, the 1-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In other embodiments, the 5-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In other embodiments, the 10-year survival rate of treated subjects isincreased by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55% about 60%,about 65%, about 70%, about 75%, about 80%, about 90%, about 95%, about100%, or any range bound by these values.

In yet other embodiments, the subject has a sustained remission of atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, at least 11 months, at least 12 months, at least 14months, at least 16 months, at least 18 months, at least 20 months, atleast 22 months, at least 24 months, at least 27 months, at least 30months, at least 33 months, at least 36 months, at least 42 months, atleast 48 months, at least 54 months, or at least 60 months or more.

In other embodiments, the method may help to treat or alleviateconditions, symptoms, or disorders related to cancer. In someembodiments, these conditions or symptoms may include, but are notlimited to, anemia, asthenia, cachexia, Cushing's Syndrome, fatigue,gout, gum disease, hematuria, hypercalcemia, hypothyroidism, internalbleeding, hair loss, mesothelioma, nausea, night sweats, neutropenia,paraneoplastic syndromes, pleuritis, polymyalgia rheumatica,rhabdomyolysis, stress, swollen lymph nodes, thrombocytopenia, Vitamin Ddeficiency, or weight loss. In other embodiments, the administration ofboth the RARα agonist and CAR-modified immune cells prolongs thesurvival of the individual being treated relative to treatment with theCAR-modified immune cells alone.

LIST OF PARTICULAR EMBODIMENTS

The following listing of embodiments is illustrative of the variety ofembodiments with respect to breadth, combinations and sub-combinations,class of invention, etc., elucidated herein, but is not intended to bean exhaustive enumeration of all embodiments finding support herein.

Embodiment 1

A method of cancer immunotherapy comprising administering to a subjectin need thereof chimeric antigen receptor-modified immune cells(CAR-MIC) and at least one differentiating RAR active agent.

Embodiment 2

A method of treating cancer comprising administering to a subject inneed thereof (CAR-MIC) and at least one differentiating RAR activeagent.

Embodiment 3

A method of potentiating CAR-MIC cancer immunotherapy comprisingadministering at least one differentiating RAR active agent to a cancerpatient who is receiving, has received, or is scheduled to receiveCAR-MIC.

Embodiment 4

A method of cancer immunotherapy comprising administering to a subjectin need thereof, a differentiating RAR active agent and CAR-MIC, whereinthe CAR-MIC are cultured in a culture medium comprising at least oneimmunomodulatory RAR/RXR active agent prior to being administered to thesubject.

Embodiment 5

A method of prolonging the disease-free survival of a cancer patientcomprising administering CAR-MIC and at least one differentiating RARactive agent.

Embodiment 6

A method of decreasing toxicity of CAR-MIC therapy comprisingadministering to a subject in need thereof at least one differentiatingRAR active agent in combination with the CAR-MIC such that as a resultof the combination, a lower dose of CAR-MIC are administered than if theCAR-MIC were administered alone.

Embodiment 7

The method of Embodiment 6, wherein the efficacy is the same orincreased as compared to the administration of CAR-MIC alone.

Embodiment 8

A method of increasing efficacy of CAR-MIC therapy comprisingadministering to a subject in need thereof at least one differentiatingRAR active agent in combination with the CAR-MIC such that as a resultof the combination, a higher dose of CAR-MIC are administered than ifthe CAR-MIC were administered alone.

Embodiment 9

The method of Embodiment 8, wherein the toxicity is the same ordecreased as compared to the administration of CAR-MIC alone.

Embodiment 10

The method of any one of Embodiments 1-9, further comprisingadministration of an immune checkpoint inhibitor.

Embodiment 11

The method of Embodiment 10 wherein the immune checkpoint inhibitor isan inhibitor of at least one of CTLA-4, PD-1, TIM-3, LAG-3, PD-L1ligand, B7-H3, B7-H4, BTLA, or is an ICOS, or OX40 agonist.

Embodiment 12

The method of Embodiment 10 or 11, wherein the immune checkpointinhibitor is an antibody.

Embodiment 13

The method of any one of Embodiments 1-12, wherein the differentiatingRAR active agent is a RARα agonist.

Embodiment 14

The method of Embodiment 12, wherein the RARα agonist isCYP26-resistant.

Embodiment 15

The method of Embodiment 12 or 13, wherein the RARα agonist is a RARαselective agonist.

Embodiment 16

The method of any one of Embodiments 13-15, wherein the RARα agonist isa compound of general formula (V):

wherein R¹ is H or C₁₋₆ alky, R² and R³ are independently H or F, and R⁴is a halogen.

Embodiment 17

The method of Embodiment 16, wherein R⁴ is F, CL, BR, or I.

Embodiment 18

The method of any one of Embodiments 13-15, wherein the RARα agonist isa compound of general formula (VI):

wherein R¹ is H or C₁₋₆ alkyl.

Embodiment 19

The method of any one of Embodiments 13-15, wherein the RARα agonist isa compound of general formula (III):

Embodiment 20

The method of any one of Embodiments 13-14, wherein the RARα agonist istamibarotene (AM80;4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benzoicacid).

Embodiment 21

The method of any one of Embodiments 13-14, wherein the RARα agonist isAM580(4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid).

Embodiment 22

The method of any one of Embodiments 13-14, wherein the RARα agonist isRe 80(4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid).

Embodiment 23

The method of any one of Embodiments 1-22, wherein the differentiatingRAR active agent or RARα agonist is administered daily.

Embodiment 24

The method of any one of Embodiments 1-22, wherein the differentiatingRAR active agent or RARα agonist is administered twice a day.

Embodiment 25

The method of Embodiment 23 or 24, wherein the daily dosage of RARactive agent or RARα agonist is in a range of from about 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 mg/m²/day, to about15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 mg/m²/day, the endpoints of the range selected so that the low endof the range is less than or equal to the high end of the range.

Embodiment 26

The method of any one of Embodiments 1-25, further comprisingadministering at least one cancer chemotherapy or targeted therapyagent.

Embodiment 27

The method of Embodiment 26, wherein the at least one cancerchemotherapy or targeted therapy agent is bortezomib.

Embodiment 28

The method of any one of Embodiments 1-26, wherein the subject or cancerpatient has a hematologic malignancy.

Embodiment 29

The method of Embodiment 28, wherein the hematologic malignancy isselected from acute myeloid leukemia, chronic myelogenous leukemia(CML), including accelerated CML and CML blast phase, acutelymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's disease,non-Hodgkin's lymphoma, including follicular lymphoma and mantle celllymphoma, B-cell lymphoma, T-cell lymphoma, multiple myeloma,Waldenstrom's macroglobulinemia, myelodysplastic syndromes, includingrefractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts (RAEB), and RAEB in transformation,and myeloproliferative syndromes.

Embodiment 30

The method of Embodiment 28 or 29, wherein the hematologic malignancy isa leukemia.

Embodiment 32

The method of Embodiment 28 or 29, wherein the hematologic malignancy ismultiple myeloma.

Embodiment 33

The method of any one of Embodiments 1-26, wherein the subject or cancerpatient has a solid tumor.

Embodiment 34

The method of Embodiment 33, wherein the pancreatic cancer, bladdercancer, colorectal cancer, breast cancer, prostate cancer (e.g.,androgen-dependent and androgen-independent prostate cancer), renalcancer, hepatocellular cancer, lung cancer (e.g., non-small cell lungcancer (NSCLC), small cell lung cancer (SCLC), bronchoalveolar carcinoma(BAC), and adenocarcinoma of the lung), ovarian cancer (e.g.,progressive epithelial or primary peritoneal cancer), cervical cancer,gastric cancer, esophageal cancer, head and neck cancer (e.g., squamouscell carcinoma of the head and neck), melanoma, neuroendocrine cancer,brain tumors (e.g., glioma, anaplastic oligodendroglioma, adultglioblastoma multiforme, and adult anaplastic astrocytoma), bone cancer,and soft tissue sarcoma.

Embodiment 35

The method of any one of Embodiments 1-34, wherein the differentiatingRAR active agent or RARα agonist is administered for at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 1 week, at least 2 weeks, at least 3 weeks, or at least one month,before administration of CAR-MIC.

Embodiment 36

The method of Embodiment 35, wherein administration of thedifferentiating RAR active agent or RARα agonist is discontinued 2 to 10days prior to administration of CAR-MIC.

Embodiment 37

The method of Embodiment 35, wherein administration of thedifferentiating RAR active agent or RARα agonist is discontinued at apoint in an interval from 2 days prior to 2 days subsequent toadministration of CAR-MIC.

Embodiment 38

The method of Embodiment 35, wherein administration of thedifferentiating RAR active agent or RARα agonist is continued for aninterval subsequent to administration of CAR-MIC greater than 2 days.

Embodiment 39

The method of Embodiment 38, wherein administration of thedifferentiating RAR active agent or RARα agonist is discontinued 14 dayssubsequent to administration of CAR-MIC.

Embodiment 40

The method of any one of Embodiments 35-39, wherein the differentiatingRAR active agent or RARα agonist is administered for a period of timebeginning 4 weeks before administration of CAR-MIC is scheduled tobegin.

Embodiment 41

A method of treatment constituting multiple cycles of treatment, whereinany one of Embodiments 35-40 constitutes one cycle.

Embodiment 42

The method of any one of Embodiments 35-41, in which administration ofthe differentiating RAR active agent or RARα agonist having beendiscontinued, wherein administration of the differentiating RAR activeagent or RARα agonist is resumed prior to a scheduled furtheradministration of CAR-MIC.

Embodiment 43

The method of Embodiment 42 wherein the differentiating RAR active agentor RARα agonist is administered for at least 2 days, at least 3 days, atleast 4 days, at least 5 days, at least 6 days, at least 1 week, atleast 2 weeks, at least 3 weeks, or at least one month, beforeadministration of the CAR-MIC.

Embodiment 44

The method of any one of Embodiments 41-43, wherein cycles of treatmentcontinue for at least 6 months following 1st administration of theCAR-MIC.

Embodiment 45

The method of any one of Embodiments 41-43, wherein cycles of treatmentcontinue until a durable complete response is obtained.

Embodiment 46

The method of any one of Embodiments 41-43, wherein cycles of treatmentcontinue as long as there is continued tumor regression.

Embodiment 47

The method of any one of Embodiments 41-43, wherein cycles of treatmentcontinue as long as there is stable disease or the cancer does notprogress.

Embodiment 48

The method of Embodiment 45 or 47, wherein treatment is suspendedfollowing the attainment of complete response or stable disease andcycles of treatment resume upon disease progression.

Embodiment 49

The method of any one of Embodiments 1-48, wherein the CAR-MIC areadministered by intravenous injection or infusion.

Embodiment 50

The method of any one of Embodiments 1-48, wherein the CAR-MIC areadministered by intratumoral injection.

Embodiment 51

The method of any one of Embodiments 1-50, wherein 1×10⁶ to 3×10¹⁰ cellsare administered.

Embodiment 52

The method of Embodiment 1-51, wherein 1×10⁵ to 1×10⁸ cells per kilogramof patient body weight are administered.

Embodiment 53

The method of any one of Embodiments 1-52, wherein the CAR-MIC is aCAR-T cell.

Embodiment 54

The method of any one of Embodiments 1-52, wherein the CAR-MIC is aCAR-NKT cell.

Embodiment 55

The method of any one of Embodiments 1-52, wherein the CAR-MIC is aCAR-macrophage.

Embodiment 56

A differentiating RAR active agent for use in reducing the toxicity ofCAR-MIC therapy.

Embodiment 57

A differentiating RAR active agent for use in potentiating theimmunotherapeutic effect of CAR-MIC in the treatment of cancer.

Embodiment 58

CAR-MIC and a differentiating RAR active agent for use in treatingcancer.

Embodiment 59

CAR-MIC and a differentiating RAR active agent for use in cancerimmunotherapy.

Embodiment 60

The differentiating RAR active agent for use according to Embodiment 56or 57, or the CAR-MIC and a differentiating RAR active agent for useaccording to Embodiment 58 or 59, in a cancer patient who is receiving,has received, or is scheduled to receive, CAR-MIC.

Embodiment 61

Use of a differentiating RAR active agent in the manufacture of amedicament for potentiating the immunotherapeutic effect of CAR-MIC inthe treatment of cancer.

Embodiment 62

Use of CAR-MIC and a differentiating RAR active agent in the manufactureof medicaments for cancer immunotherapy.

Embodiment 63

Use of CAR-MIC and a differentiating RAR active agent in the manufactureof medicaments for prolonging the disease-free survival of a cancerpatient.

Embodiment 64

Use of a differentiating RAR active agent in the manufacture of amedicament for reducing the toxicity of CAR-MIC therapy.

Embodiment 65

Use of CAR-MIC and a differentiating RAR active agent in the manufactureof medicaments for treating cancer.

Embodiment 66

The use of any one of embodiments 61-65, wherein the differentiating RARactive agent medicament is for use in a cancer patient who is receiving,has received, or is scheduled to receive, CAR-MIC.

It should be manifest that each of Embodiments 56-66 can be modified ina manner similar to the modification of Embodiments 1-6 by embodiments7-55.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification.

Example 1 Binding of Test Compounds to RAR Receptors and Activation ofReporter Genes

Retinoic acid receptor transactivation activity and binding efficienciesare determined essentially as described in U.S. Pat. Nos. 5,298,429 and5,071,773, incorporated by reference herein. Transactivation assaysemploy expression plasmids encoding the full length receptors RARα,RARβ, and RARγ. Reporter plasmids containing the herpes virus thymidinekinase promoter and the appropriate retinoic acid receptor responseelement (RAREs) are positioned upstream of an open coding regionencoding firefly luciferase.

Binding assays are performed using a classic competition assay format inwhich cloned receptor RAR molecules are first loaded with radiolabeledall-trans-retinoic acid (RAR) and then the amount of radioactivityliberated with increasing concentration of test compound is measured.

The assays are used to identify RARα agonists as disclosed herein above.

Example 2 Compound IRX5183 is RARα Specific

To determine whether the compounds having a structure of formula I areRARα selective agonists, the compound IRX5183 was examined for itsability to bind to RARα, RARβ, and RARγ using a displacement assay tomeasure agonist binding affinity and a transactivation assay to measureagonist activity (described in U.S. Pat. No. 5,455,265, which is herebyincorporated by reference). These results indicate that compound IRX5183selectively binds to RARα with high affinity (Table 1) and specificallyactivates RARα (FIG. 1. Such a RARα selective agonist could minimize theadverse effects related to pan-activation including mucocutaneoustoxicity, headache, and proinflammatory events in clinical studies.

TABLE 1 IRX5183 Binding Affinities for RARα, RARβ, and RARγ RARα RARβRARγ 4.7 nM >10,000 nM >10,000 nM

Example 3 RARα Signaling Induces Foxp3 Expression

To determine which of the RAR (RARα, RARβ, RARγ) signaling pathways isinvolved in the induction of Foxp3 expression, naive CD4⁺ CD25⁻ FoxP3⁻cells were purified from a Foxp3-GFP mouse using flow cytometry bysorting and isolating based upon a GFP⁻ phenotype. These cells wereactivated polyclonally with αCD3 in vitro in the presence of IL-2 andTGF-β. To identify the RAR involved in RA-induced Foxp3 expression, thecultured cells were incubated with RAR selective agonists. The culturedcells were then scored for the frequency of GFP⁺ (Foxp3⁺). With respectto the use of selective agonists, only the RARα agonist exertedsignificant impact on the expression of Foxp3 inducing nearly 100%Foxp3+ T cells, with enhancement on the expression of α4β7 and CCR9 (guthoming receptors) (FIG. 2). The RARγ and RARβ agonists were withouteffect.

Example 4 RARα Selective Agonists Regulates T Cell Differentiation

To determine whether a RARα agonist could affect T cell differentiation,T cells were incubated with a RARα agonist to determine its effect onFoxp3 expression. Naive CD4⁺ CD25⁻ FoxP3⁻ cells were purified from aFoxp3-GFP mouse using flow cytometry by sorting and isolating based upona GFP⁻ phenotype. These cells were activated polyclonally with αCD3 invitro in the presence of IL-2 and TGF-β. These cells were then culturedin media with various concentrations of compound IRX5183 (a RARαagonist) and the expression of FoxP3-GFP was analyzed by flow cytometry.The RARα agonist compound IRX5183 enhanced differentiation ofimmunosuppressive Treg cells and inhibited differentiation ofinflammatory TH17 cells from naïve T cells in vitro (Table 2).

TABLE 2 RARα agonist Effects on T Cell Differentiation Treg cell Th17cell Percent Percent Concentra- Differentia- Concentra- Differentia-RARα tion tion tion tion agonist (nM) (%) (nM) (%) Compound 0 25 0 32IRX5183 0.1 26 0.1 32 1 55 1 21 10 90 10 11 100 ND 100 5

To expand on the finding above, the in vivo effects of a RARα agonist onT cell differentiation was evaluated in a mouse model. Mice were treatedwith 100 μg of compound IRX5183 or an equivalent volume of DMSO (vehiclecontrol) every other day for 10 days. Lymphocytes from the blood andspleen were then isolated and FoxP3 expression in CD4⁺ T cells wasassessed. The data shows that following administration of compoundIRX5183 there was a significant increase in the percentage of Foxp3⁺ Tcells in the spleen and blood of treated mice (Table 3).

TABLE 3 RARα Agonist Effects on T Cell Differentiation Foxp3+ Expression(%) Tissue DMSO IRX5183 Blood 2.4 4.3 Spleen 10 25

Conversely, AGN196996, an RARα selective antagonist, increases Th17 cellnumbers and decreases Treg cell numbers in the above in vitro and invivo assays (data not shown).

Example 5 The Bone Marrow Niche Induces a Bortezomib Resistance inMultiple Myeloma

Multiple myeloma (MM) is characterized by the proliferation of malignantplasma cells (PCs) within the BM and their production of monoclonalimmunoglobulin (Ig). Novel therapies, including proteasome inhibitors,have significantly extended the survival of patients with MM but havefailed to achieve a cure. Increasing evidence demonstrates thatinteractions with the BM microenvironment play a critical role in thesurvival of MM cells during chemotherapy. However, the mechanismsmediating this BM niche-dependent chemoprotection are incompletelyunderstood and remain a critical area of research.

Certain MM cells that resemble mature B cells and are resistant tobortezomib (BTZ). Like their normal B cell counterparts, these CD138− MMcells are capable of clonogenic growth and differentiation into CD138+PCs. Moreover, these cells are enriched during minimal residual disease(MRD), suggesting a critical role in disease relapse. Differential BTZsensitivity of CD138+ and CD138− MM cells may be explained by theirsecretory activity. As a result of their abundant Ig production, CD138+PCs are highly dependent on an intact proteasome pathway to degradeimproperly folded proteins. Conditions that disrupt protein degradationby the proteasome activate a cellular stress pathway known as theunfolded protein response (UPR), which counteracts ER stress bydecreasing protein synthesis and promoting protein degradation. Ifhomeostasis cannot be reestablished, UPR activation eventually leads toapoptosis. On the other hand, CD138− MM cells exhibit limited Igproduction and low ER stress and are less dependent onproteasome-mediated degradation of misfolded proteins.

Previous studies demonstrated that BM stromal cells induce an immaturedrug-resistant phenotype in MM. BM stroma creates a retinoic acid-low(RA-low) environment via CYP26 that prevents the differentiation ofnormal and malignant cells. Since retinoid signaling promotes PCdifferentiation and Ig production, this study determined whether the BMniche via stromal CYP26 activity induces BTZ resistance by preventing PCdifferentiation (Alonso S. et al., J. Clin. Invest. 126:4460-4468,2016). An RA-low environment induced by stromal CYP26 is responsible formaintaining a B cell-like, BTZ-resistant phenotype in MM cells. Directlyinhibiting CYP26 or bypassing stromal protection via a CYP26-resistantretinoid rescues PC differentiation and BTZ sensitivity. Furthermore, wedescribe a bidirectional crosstalk, in which paracrine Hedgehog secretedby MM cells reinforces a protective niche via an increase in the abilityof BM stroma to inactivate RA. These data indicate that modulation of RAsignaling is an attractive therapeutic strategy for overcoming BTZresistance in the MM BM microenvironment.

Methods

Cell Cultures.

All cell lines were purchased from the American Type Culture Collection.H929, MM.1S, and U266 cells were cultured in RPMI 1640 with 10% FCS(Fetal Calf Serum), 2 mM L-glutamine, and 100 μg/mlpenicillin-streptomycin (P/S). OP-9 cells were cultured in α-MEM, 20%FCS, L-glutamine, and P/S. Cell lines were authenticated by short-tandemrepeat profiling.

Primary MM cells were obtained from patients with newly diagnosed orrelapsed MM under an IRB-approved protocol. Briefly, mononuclear cellswere isolated from fresh BM aspirates by density gradient centrifugation(Ficoll-Paque); CD138+ cells were then selected via magnetic beads andcolumns and incubated in RPMI 1640, 10% FCS, L-glutamine, and P/S at 37°C.

Primary human BM stromal cells were derived from aspirates collectedfrom healthy donors under an IRB-approved protocol. Briefly, totalmononuclear cells isolated from BM aspirates were cultured in Iscove'smodified Dulbecco's medium (IMDM) supplemented with 10% horse serum, 10%FCS, 10⁻⁵ M hydrocortisone 21-hemisuccinate, P/S, and 0.1 mMβ-mercaptoethanol (β-ME) (FBMD1 media). The following day, cells insuspension were removed by washing twice with PBS, and the media werereplaced. Attached stromal cells were incubated at 33° C. until aconfluent monolayer was obtained. Mouse primary BM stromal cells wereisolated following the same protocol, after isolation of total BMmononuclear cells from mouse femurs.

Vectors and Viral Supernatants.

To generate Smo-KO and WT stroma, BM stromal cells were derived fromSmo^(fl/fl) mice and transduced with the retroviral vector PIG-Creencoding Cre-recombinase (Addgene; catalog 50935) or a control vector(Addgene; catalog 18751), respectively. Successfully infected cells wereselected using 4 μg/ml puromycin for 5 days and confirmed by expressionof GFP via flow cytometry. The pLenti-CMV-LUC-Puro lentiviral vector(plasmid 17477) was used to generate H929 Luc+ cells.

To generate CYP26A1-overexpressing stromal cells, WT and Smo-KO stromalcells were transduced with the lentiviral vector pBABE-neo (Addgene;catalog 1767) that had been engineered to encode CYP26A1. Briefly,Cyp26a1 cDNA (Origene) was amplified via PCR using primers incorporatingthe restriction sites BamHI and EcoRI and cloned into the pCR2.1 vector.Cyp26a1 cDNA was confirmed via Sanger sequencing, and the fragment wasisolated after digestion with the restriction enzymes BamHI and EcoRIand subcloned into the corresponding sites of the pBABE vector.Lentiviral particles were produced as previously described. Successfullyinfected stromal cells were selected using 3 μg/ml G-418 for 10 days,and expression of Cyp26a1 was confirmed by qRT-PCR.

Coculture Experiments.

24-well plates were coated with 0.1% gelatin in PBS for 30 min at 37° C.The gelatin solution was removed, and the stromal cells were culturedovernight at a density of 5×10⁴ cells/well to obtain a confluentmonolayer. At that time, MM cell lines or primary MM cells (1×10⁵ in 2ml) were added to the stroma cultures. The stroma cocultures wereincubated at 37° C. in RPMI containing 10% FCS, L-glutamine, and P/S,with or without AGN194310 (1 μM for 5 days), R115866 (1 μM for 5 days),IRX5183 (1 μM for 5 days), or BTZ (2.5 nM for 48 hr).

Transwell Experiments.

For Transwell experiments, 6-well plates were coated with 0.1% gelatinin PBS for 30 min at 37° C. The gelatin solution was removed, and thestromal cells were cultured overnight in FBMD1 media at a density of10×10⁴ cells/well in 2 ml of media to obtain a confluent monolayer. Atthat time, Transwell inserts (Corning) were placed over the stromacultures, and MM cell lines (1×10⁶ in 1 ml) were seeded in the Transwellfor 24 hr at 37° C. Following this incubation, Transwell and MM cellswere removed, and stromal cells were detached from the wells andanalyzed by qRT-PCR for CYP26 expression.

Mobilization Experiments.

MM cells were separated from BM stromal cells by gently pipettingseveral times around the well. Detached cells were centrifuged,resuspended in fresh media, and incubated in a 24-well plate for 1 hr at37° C. During this short incubation period, contaminating stromal cellsattached to the well, while MM cells remained in suspension. MM cellswere then recovered by gently pipetting. This protocol was used forqRT-PCR and CFU coculture experiments. The purity achieved using thisprotocol was confirmed by flow cytometry to be 98%-99% MM cells and lessthan 2% contaminating stroma.

Clonogenic Assays.

After treatment, MM cells were collected, washed with PBS, and plated ata density of 5,000 cells/ml in 1 ml of 1.32% methylcellulosesupplemented with 30% FBS, 10% BSA, L-glutamine, P/S, and 0.1 mM β-ME.Cells were plated in triplicate in 35-mm culture dishes, incubated at37° C., and scored for the presence of colonies 14 days later.

qRT-PCR.

Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) according tothe manufacturer's instructions. cDNA was synthesized by reversetranscription using the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCRwas performed with iTaq SYBR Green Supermix (Bio-Rad) using sequencespecific primers. Gene expression was normalized to GAPDH, and relativequantification was calculated using ΔΔCt. All experiments were performedin duplicate and run on the Bio-Rad CFX96 machine.

Flow Cytometry.

Following treatment, MM cells were collected, washed with PBS, andstained for 15 min at room temperature with phycoerythrin-conjugated(PE-conjugated) anti-CD138. Cells were washed to remove unbound antibodyand evaluated in a FACSCalibur system (BD Biosciences). Stromal cellswere identified by GFP expression, and viable cells were identifiedusing 7-aminoactinomycin D (7-AAD). To calculate cell numbers, live GFP−cells were normalized to calibration beads.

Mouse Xenografts.

1×10⁶ H929 Luc+ cells and 1×10⁶ mouse BM stromal cells were resuspendedin 100 μl MATRIGEL®, diluted with RPMI (1:1), and injectedsubcutaneously into 16-week-old male NSG mice. After 4 days, treatmentwith BTZ (0.5 mg/kg i.p. twice weekly) and IRX (10 mg/kg i.p. daily) wasinitiated. Tumor burden was assessed by bioluminescence using the InVivo Imaging System (PerkinElmer). For imaging, mice were exposed to 120mg/kg D-luciferin via intraperitoneal injection 10-5 min before imagingand were anesthetized using isoflurane. Images were analyzed with LivingImage Software, version 2.5 (PerkinElmer), and data were quantified asphotons/second.

For the systemic MM model, 2×10⁶ Luc+/GFP+ H929 cells were injected viathe tail vein into 16-week-old male NSG mice. After engraftment, asdetermined by an exponential increase in bioluminescence, mice weretreated with BTZ (0.5 mg/kg i.p.) twice weekly and with IRX (10 mg/kg)once daily. Tumor burden was assessed by bioluminescence, as above.

Statistics.

First evaluated was whether the treatment groups were different from thecontrols using 1-way ANOVA. If the ANOVA test yielded a statisticallysignificant result, then the difference between the control group andeach treatment group was evaluated, with the P values adjusted formultiple comparisons using Dunnett's test. For experiments in which only2 sets of data were analyzed, statistical significance was evaluatedusing an unpaired, 2-tailed Student's t test. Pearson's R value forcorrelation and P values were calculated using GraphPad Prism 7(GraphPad Software).

Results

The BM microenvironment (also called “niche”) limits PC differentiationby modulating retinoid signaling. A population of MM progenitors,phenotypically similar to B cells, is intrinsically resistant to BTZ andcontributes to MRD and relapse. To investigate whether the BM nicheplays a role in determining the phenotype of MM cells, the mRNAexpression of B cell and PC markers in MM H929 cell lines (FIG. 3A-D)and MM CD138+ primary cells (FIG. 3E-H) was analyzed followingco-culture with mouse BM stroma using human-specific primers. B celllymphoma 6 (BCL6), a transcriptional repressor that promotesself-renewal of germinal center B cells and prevents PC differentiation,was upregulated in the presence of BM stromal cells (FIG. 3A, 3E). Incontrast, co-culture of MM cells with BM stroma decreased the mRNAexpression of B lymphocyte-induced maturation protein 1 (BLIMP1) andspliced X box-binding protein 1 (XBP1s) (FIG. 3B, C, F, G), which arecritical mediators of PC differentiation. Similarly, C/EBP homologousprotein (CHOP), a key component of the UPR pathway, was downregulated inthe presence of BM stromal cells (FIG. 3D, H).

The BM niche regulates hematopoietic stem cell (HSC) differentiation byexpressing the retinoid-inactivating enzyme CYP26. CYP26 enzymes werehighly expressed in BM mesenchymal cells, while their expression wasbarely detectable in MM cells. Since retinoid signaling promotes PCdifferentiation and potentiates Ig secretion, it was determined whetherstromal CYP26 is responsible for inducing a B cell phenotype in MMcells. To this end, co-culture conditions were treated with the CYP26inhibitor R115866 (R115) or the CYP26-resistant RA receptor α-selective(RARα-selective) retinoid IRX5183 (IRX). Incubation of stromaco-cultures with either R115 or IRX restored all markers to levelscomparable to those of liquid control conditions (FIG. 3A-H). Moreover,treatment of MM cells with the pan-RAR antagonist AGN194310 (AGN)mimicked the changes induced by BM stromal cells (FIG. 3A-H), limitingPC differentiation.

Expression of CD138 is a hallmark of normal PC differentiation as wellMM PCs. Consistent with mRNA levels of PC markers, surface CD138expression was markedly decreased by co-culture with BM stromal cells orincubation with AGN. Incubation of BM stromal cell co-cultures with R115or IRX restored CD138 expression in MM cells. R115 did not significantlyaffect the expression of differentiation markers in liquid conditions byquantitative reverse transcription-PCR (qRT-PCR) or flow cytometry,while IRX induced comparable changes, irrespective of the presence orabsence of BM stroma. Taken together, these data suggest that retinoidsignaling promotes PC differentiation of MM cells and that this processis blocked by stromal CYP26-mediated metabolism of RA.

A RA-Low Microenvironment Induces BTZ Resistance.

To determine whether decreased retinoid signaling contributes to BTZresistance within the BM niche, MM cell lines and MM CD138+ primarycells were incubated with BM stroma for 5 days, followed by BTZtreatment. In the absence of BM stroma (liquid), MM cells were highlysensitive to BTZ (FIG. 4A-B). However, incubation with BM stroma inducedBTZ resistance, which was overcome by CYP26 inhibition via R115 or bythe CYP26-resistant retinoid IRX. Moreover, treatment of MM cells withthe pan-RAR antagonist AGN mimicked the changes induced by BM stromalcells (FIG. 5), decreasing BTZ sensitivity.

Strategies to overcome microenvironment-dependent chemoprotection havefocused on mobilization of cancer cells from the BM niche into theperipheral circulation. It was analyzed whether the change in phenotypeand subsequent BTZ resistance of MM cells were lost upon separation fromthe BM stroma, a process that mimics mobilization. To this end, H929cells were separated from BM mesenchymal cells following a 5-day stromaco-culture, incubated in fresh media (RPMI with 10% FBS) for 0 to 48 hr,and then treated with BTZ. Interestingly, MM cells remained partiallyresistant to BTZ for up to 48 hr following detachment from stroma (FIG.5). Moreover, treatment of the co-culture conditions with R115 preventedthe development of a BTZ-resistant phenotype (FIG. 5). Thus,microenvironment-dependent BTZ resistance induced by the change in MMcell phenotype may not immediately be reversed by tumor mobilization.

To test whether retinoids can enhance BTZ activity in MM, a systemic MMxenograft was developed by injecting 2×10⁶ H929 luciferase+ (Luc+) cellsvia the tail vein of non-obese, diabetic, severe combinedimmunodeficiency IL-2 receptor γ-KO (NSG) mice. The animals wererandomized to receive IRX, BTZ, or a combination of both, and diseaseburden was followed weekly by bioluminescence imaging (FIG. 6). Micetreated with BTZ showed decreased tumor growth compared with untreatedcontrols; however, some MM cells remained resistant to BTZ, asdemonstrated by the continued increase in bioluminescence. Similarly,mice treated with IRX monotherapy showed a decrease in tumor burdencompared with untreated mice. Most important, IRX sensitized MM cells toBTZ, leading to a significant (P<0.01) decrease in disease burden.Collectively, these data suggest that an RA-low microenvironment createdby stromal CYP26 induces a BTZ-resistant phenotype, which is maintainedeven after displacement from the BM niche.

MM Cells Induce Stromal CYP26.

Recent studies have demonstrated the existence of a bi-directionalcrosstalk, in which not only stromal cells provide a protectivemicroenvironment, but also cancer cells actively adapt and build areinforced niche. Thus, it was determined whether MM cells reinforce aprotective microenvironment by strengthening the ability of BM stroma toinactivate retinoids. Stromal CYP26 expression was analyzed by qRT-PCRin BM mesenchymal cells following a 24-hr coculture with MM cells. Theisoenzyme CYP26A1 was highly upregulated by all 3 MM cell lines tested(FIG. 7A-C). In contrast, the isoenzyme CYP26B1 showed little to nochanges in mRNA levels. Conditioned media derived from MM cells alsoupregulated CYP26A1 in BM stromal cells, although to a lesser extent.This could be explained by the presence of physical interactions incoculture experiments, or the lack of continuous production of solubleligands by MM cells in conditioned media experiments. Consistent withthe latter, stromal CYP26A1 was highly upregulated when MM and BMstromal cells were separated by a Transwell that prevented physicalcontact but allowed the diffusion of soluble factors (FIG. 7A-C).

MM cells produce a variety of soluble factors including cytokines (IL-1,IL-3, IL-6, TNF-α) as well as Hedgehog ligands such as sonic hedgehog(SHH), which could impact the BM stromal compartment. Therefore, it wasdetermined whether any of these factors was responsible for the observedupregulation of CYP26A1 on BM stromal cells. Of the soluble factorstested, only SHH produced a sustained overexpression of CYP26A1, whileIL-1, IL-3, IL-6, and TNF-α had no significant effects. Whereas SHH isexpressed by BM stromal cells and thus may be able to activate theHedgehog pathway in an autocrine manner, its expression was considerablyhigher in MM cells compared with that detected in BM stroma, suggestingthat paracrine activation may play a dominant role. Consistent withthis, there was a statistically significant correlation between the mRNAlevels of SHH in MM cells and activation of stromal Hedgehog signalingas determined by protein patched homolog 1 (PTCH1) expression. Moreover,activation of stromal Hedgehog significantly correlated with CYP26A1upregulation. Specifically, MM.1S cells with the highest expression ofSHH also induced the highest expression of both PTCH1 and CY26A1 instromal cells. SHH has a half-life of less than 1 hr, which may explainthe reduced effect of MM-conditioned media on stromal CYP26A1 expressioncompared with that observed in coculture and Transwell experiments.

To confirm the role of paracrine Hedgehog on this interaction,smoothened (Smo), a membrane receptor that transduces SHH signaling, wasknocked out at the genomic level in the mesenchymal compartment. Forthis, BM mesenchymal cells derived from Smo^(fl/fl) mice were transducedwith a retroviral vector encoding Cre recombinase (Smo-KO stroma). MouseSmo^(fl/fl) stromal cells transduced with an empty retroviral vectorwere used as a control (WT stroma). The transduced BM stromal cells werecocultured with MM cells for 24 hr. As expected, Smo-KO stroma had adecreased ability to upregulate Cyp26a1 in response to MM cells comparedwith WT stroma (FIG. 8A-C). Similarly, the SMO inhibitor cyclopaminepartially overcame stromal Cyp26a1 upregulation by MM cells. These datasuggest that MM cells modulate stromal CYP26 expression at least in partvia paracrine SHH.

Paracrine Hedgehog Produced by MM Cells Reinforces a ProtectiveMicroenvironment.

Given the observations that stromal CYP26 activity may be responsiblefor BTZ resistance in MM cells, it was assessed whether paracrineHedgehog secreted by MM cells reinforces a chemoprotective niche byregulating retinoid metabolism. It was first investigated whethermodulation of Hedgehog signaling paralleled the retinoid-dependentphenotypes observed previously. Disruption of paracrine Hedgehogsignaling in Smo-KO stroma cocultures partially restored PCdifferentiation (downregulation of BCL6 and upregulation of BLIMP1,XBP1, and CHOP) in H929 (FIG. 11A-D) and primary CD138+ MM cells (datanot shown). Surface expression of CD138 was also restored in thepresence of Smo-KO stroma. As expected, these findings were associatedwith an increased sensitivity to BTZ of MM cells treated in the presenceof Smo-KO stroma compared with WT stroma.

To demonstrate that paracrine Hedgehog indeed induces a BTZ-resistantphenotype by increasing the ability of BM stroma to inactivateretinoids, Cyp26a1 expression in Smo-KO stroma was rescued vialentivirus-mediated gene transfer (pBABE-Cyp26a1) in order to achievecomparable CYP26A1 levels in WT (WT-Cyp26a1) and Smo-KO (Smo-KO-Cyp26a1)stromal cells. If the role of paracrine Hedgehog was independent ofretinoid signaling, the relative inability of Smo-KO stroma to induce aB cell phenotype and BTZ resistance should have persisted even afterCyp26a1 upregulation. However, Cyp26a1 overexpression rescued theability of Smo-KO stroma to induce a B cell phenotype and restored theexpression of differentiation markers and BTZ resistance to levelscomparable to those detected in WT and WT-Cyp26a1 stroma cocultureconditions. This finding is consistent with the hypothesis thatparacrine Hedgehog reinforces a protective niche via Cyp26a1upregulation.

To study to what extent an RA-low environment created by the BM stromaand enhanced by MM cells via paracrine Hedgehog signaling contributes toBTZ resistance, a xenograft model of MM-niche interactions wasdeveloped. Each mouse carried 2 subcutaneous tumors consisting of H929Luc+ cells and either WT (anterior tumors) or Smo-KO stroma (posteriortumors). Mice were treated with IRX (10 mg/kg i.p. daily), BTZ (0.5mg/kg i.p. twice weekly), or a combination of both. The growth of tumorsbearing WT or Smo-KO stroma was not different in untreated orIRX-treated groups (FIGS. 9 and 10). Consistent with in vitro data,tumors with WT stroma were refractory to BTZ treatment, as determined byan exponential increase in bioluminescence, while tumors carrying Smo-KOstroma showed a significant response (FIG. 8). Moreover, the combinationof IRX and BTZ resulted in a significant and equivalent response,regardless of the phenotype of the stromal compartment (FIG. 10). Whilesome tumors in the treatment group receiving combined IRX and BTZappeared to have regressed completely, even after anatomical study, thiswas not the case for all the mice in this group. Flow cytometricanalyses of the tumors after treatment revealed no differences in the invivo growth of WT or Smo-KO stroma. Taken together, these data suggestthat paracrine Hedgehog secreted by MM cells modulates retinoidsignaling and BTZ sensitivity in the BM niche via CYP26A1 upregulation.

Given their high secretion of Ig, PCs are particularly sensitive toproteasome inhibition, and this accounts for the high remission ratesachieved in MM patients treated with this family of drugs. Nonetheless,BTZ has failed to achieve a cure. A population of MM cells,phenotypically similar to B cells, survive BTZ treatment and are able todifferentiate into PCs and recapitulate the original disease. Despiteefficient elimination of MM PCs, these MM B cells survive BTZ treatmentand become the predominant cell population during MRD. Consequently, newtherapeutic strategies targeting MM B cells are required. A retinoid-lowmicroenvironment created by stromal CYP26 maintained an immature,BTZ-resistant phenotype in MM. Thus, these data reveal a therapeuticopportunity to overcome BTZ resistance in the MM microenvironment usingCYP26-resistant retinoids.

Despite being extensively studied in many hematological malignancies,the use of retinoids as differentiation therapy has proved beneficialonly in patients with acute promyelocytic leukemia (APL). CYP26expression by BM stromal cells may explain the lack of a clinicalbenefit of natural retinoids, despite their in vitro activity. Recentstudies have highlighted the efficacy of CYP-resistant syntheticretinoids in differentiating cancer cells and sensitizing them totargeted therapy. For instance, AM80 differentiates FMS-like tyrosinekinase 3/internal tandem duplication (FLT3/ITD) acute myeloid leukemia(AML) cells and increase their sensitivity to FLT3 inhibitors.Similarly, synthetic retinoids reverse a stem cell phenotype inBCR-ABL1+ leukemic lymphoblasts and substantially increase theirresponsiveness to tyrosine kinase inhibitor (TKI) therapy in vivo. Suchstrategies to bypass stromal CYP26 could expand the clinicaleffectiveness of retinoid therapy.

MM cells utilize physical contacts to maintain drug resistance andsurvive within the BM niche. Thus, therapeutic strategies to overcomestromal chemoprotection have focused on mobilization of malignant cellsfrom the BM niche by targeting adhesion molecules or chemokines such asCXCR4. MM cells exposed to a retinoid-low microenvironment acquire aBTZ-resistant phenotype that is maintained even after these cells aredisplaced from their niche. Initial clinical studies have shown improvedresponse rates in relapse/refractory patients receiving the CXCR4inhibitor plerixafor in combination with BTZ; however, this data suggestthat such mobilization approaches may be insufficient to eliminate MM Bcells.

Recent studies have demonstrated the existence of a bidirectionalcommunication, in which not only stromal cells provide a chemoprotectiveniche, but also cancer cells actively shape and reinforce theirmicroenvironment. The role of paracrine Hedgehog has been studiedextensively in solid malignancies. In this system, ligands secreted bycancer cells activate the Hedgehog pathway in neighboring stromal cells,enhancing their chemoprotective properties via incompletely understoodmechanisms. This data suggest that paracrine Hedgehog may work at leastin part by increasing the ability of stroma to inactivate retinoidsthrough upregulation of CYP26 and thus to maintain a BTZ-resistantphenotype in MM. Interestingly, CYP26 upregulation is associated with an“activated stromal subtype” and a significantly worse prognosis inpatients with pancreatic cancer, a disease in which paracrine Hedgehogsignaling is well established. The extent to which Hedgehog ligandsproduced by cancer cells contribute to this “activated” stromalphenotype and high CYP26 levels is unknown. Moreover, BM mesenchymalcells migrate and become a relevant cell population in the stromalcompartment of these tumors.

The endosteal region is the primary niche of MM, AML, andmicrometastatic disease from solid tumors. Within the osteoblasticregion, these cancer cells maintain a quiescent, stem cell phenotype andare protected from chemotherapy-induced apoptosis. It is likely thatthese cancer cells rely on the same cues from the BM microenvironment asnormal hematopoietic stem cells do to survive chemotherapy andperpetuate the disease. The BM microenvironment protected MM and AMLcells by directly inactivating various chemotherapy agents viaexpression of CYP3A4 and other detoxifying enzymes. Another potentialmechanism of microenvironment-mediated drug resistance is nowdemonstrated: creation of a retinoid-low niche that maintains adrug-resistant B cell phenotype. A CYP26-resistant retinoid (IRX5183)potentiated the activity of BTZ against MM in the BM niche provides atherapeutic opportunity to bypass this mechanism of resistance.

Example 6 Phase I/II Clinical Study of IRX5183 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is successfully treated in only 30-40% ofyounger patients and very few older patients with standard chemotherapyregimens. Given the clinical activity of all-trans retinoic acid (ATRA;retinoic acid, RA) in acute promyelocytic leukemia (APL), ATRA wasconsidered an attractive therapeutic strategy for other AML subtypes.APL, and most non-APL AMLs undergo terminal differentiation and aretherefore successfully treated by ATRA in vitro. However, ATRA has notproven effective in non-APL AMLs in clinical trials.

Retinoic acid (RA) plays a significant role in the differentiation ofhematopoietic stem cells (HSCs). The cytochrome P450 enzyme CYP26,expressed in bone marrow (BM) stromal cells, inactivates RA, therebylimiting differentiation of HSCs. Several AML cell lines, both APL andnon-APL, are sensitive to RA-induced differentiation, but this effectwas abrogated in the presence of BM stroma. Thus, it may be useful totreat AML with a retinoid that is resistant to metabolism by the CYP26pathway. IRX5183 is a RARα selective agonist which is resistant to CYP26metabolism. Use of IRX5183 in AML provides a novel targeted approach tothis disease, which has the potential to change the prognosis of thisand other hematologic malignancies. Thus, a phase I/II clinical trialwill be conducted of IRX5183 in relapsed/refractory AML.

Study Objectives

Dose Escalation Phase Primary Objectives:

-   -   Evaluate safety and toxicity associated with administration of        IRX5183 in patients with relapsed and refractory AML by        determining the dose limiting toxicities (DLT) and        maximally-tolerated dose (MTD).    -   Determine pharmacokinetic (PK) parameters of IRX5183 in the        peripheral blood.

Dose Escalation Phase Secondary Objectives:

-   -   Determine the PK parameters of IRX5183 in the bone marrow.    -   Define differentiation profiles associated with IRX5183, BM        cellular retinoid concentrations, blast counts, and cytogenetics        at different dose levels.

Dose Expansion Phase Primary Objectives:

-   -   Define differentiation markers, BM retinoid concentrations,        blast counts, and cytogenetics in AML patients at the optimal        dose level.    -   Obtain preliminary efficacy data of IRX5183 in terms of complete        response (CR), partial response (PR), and hematological        improvement (HI) in both cohorts of patients.

Dose Expansion Phase Secondary Objectives:

-   -   Define toxicity profiles of IRX5183 at the optimal dose in both        patient cohorts.    -   Obtain data on correlations between IRX5183-induced        differentiation and toxicity and clinical responses.

Eligibility Criteria—Dose Escalation/Determination.

-   -   Patients must be able to understand and voluntarily sign an        informed consent form.    -   Age 18-70 years at the time of signing the informed consent.    -   Able to adhere to the study visit schedule and other protocol        requirements.    -   Life expectancy of greater than 6 months.    -   Must have pathologically confirmed AML with one or two prior        courses of induction chemotherapy or hypomethylating agent        therapy or relapsed after complete remission, before or after        allogeneic bone marrow transplant, AND no plans for further        intensive chemotherapy.    -   Patients must not have received any other treatment for their        disease (aside from hydroxyurea for control of blast count in        AML patients), including hematopoietic growth factors, within        three weeks of beginning the trial, and should have recovered        from all toxicities of prior therapy (to grade 0 or 1).    -   ECOG performance status of ≤2 at study entry, or Karnofsky ≤60%.    -   Laboratory test results within these ranges:        -   Calculated creatinine clearance by MDRD (CrCL)>50            ml/min/1.73 squared meter        -   Total bilirubin ≤2.0 mg/dL unless due to Gilbert's syndrome,            hemolysis, or ineffective hematopoiesis AST (SGOT) and ALT            (SGPT)≤3×ULN    -   Females of childbearing potential must have negative pregnancy        test.    -   Patients must have no clinical evidence of CNS or pulmonary        leukostasis, disseminated intravascular coagulation, or CNS        leukemia.    -   Patients must have no serious or uncontrolled medical        conditions.

Eligibility Criteria—Dose Expansion.

This phase will follow the noted eligibility criteria above, includingpathologically confirmed chronic myelomonocytic leukemia (CMML) withhigh risk features at the time of referral as defined by:

-   -   INT-2 or high IPSS score    -   CMML with ≥5% marrow blasts, or RBC or platelet        transfusion-dependency, abnormal karyotype, or proliferative        features

Treatment Plan

For the dose escalating phase, IRX5183 is administered orally in dailydoses continually in 28 day cycles until toxicity or diseaseprogression. Bone marrow testing during each of the first 4 cyclesdetermines marrow status and response. The starting dose (DL1) of singleagent IRX5183 is 30 mg/m²/day, and the individual dosing levels arenoted below:

Dose level (DL) Daily dose (mg/m²) DL(−1) 15 DL1 30 DL2 45 DL3 60 DL4 75

The phase-expansion part of the study uses the optimal dose identifiedin the phase-escalation part of the study and will recruit 26 patients.

Dose levels are explored according to a traditional 3+3 design, with anaim to enroll 3 subjects at a time to determine the toxicity profile ofIRX5183 in AML patients. If none of the three patients receiving DL1experiences a DLT, another three patients will be treated at the nexthigher dose level. However, if one of the first three patientsexperiences a DLT, three more patients will be treated at the same doselevel. The dose escalation will continue until at least two patientsamong a cohort of 3-6 patients experience DLTs. If two or more patientsexperience DLT on DL1, the next patient will be recruited to DL(−1). TheMTD of single agent IRX5183 will be the highest dose at which 0 or 1 DLTare seen in a cohort of six subjects.

For the phase 2 dose expansion cohort, patients with AML are continuedto be enrolled at the MTD, with goal of enrolling 26 patients (inclusiveof patients treated at the MTD in first phase of the trial). Patientscontinue on single agent IRX5183 until they experience toxicity ordisease progression. If patients achieve a complete remission, they havethe option to consolidate with transplant, chemotherapy, and/or continueon maintenance IRX5183. If patients achieve a partial response orhematologic improvement they have the option to obtain salvage therapyin combination with IRX5183.

Pharmacokinetics Analyses.

Plasma concentrations of IRX5183 are evaluated for the escalation andexpansion phases, targeting safe and effective retinoid levels bypharmacokinetics using LCM-MS (liquid chromatography-mass spectroscopytandem). Targeting peak levels of 1 μM should avoid systemic toxicity,while presumably preserving local BM niche retinoid levels. The plasmaconcentration of IRX5183 is obtained using a single 2 mL blood sample,pre-dose on day 14. Samples are shipped to and analyzed by thedesignated analytical laboratory.

Pharmacodynamics Analyses.

In addition to assessing standard clinical response criteria, BMcellular (normal HSCs and LSCs) concentrations, peripheral blood andbone marrow blast counts, markers of differentiation, apoptosis, andclonogenic growth are determined. A bone marrow aspirate and biopsy areobtained at baseline, on day 14, and at the end each of the first 4cycles of therapy. Differentiation is assessed using flow cytometry,comparing expression of differentiation markers on CD45 positive cellsand ALDHint LSCs on day 14 marrow versus baseline. FISH analysis is alsoconducted after each cycle for patients with baseline abnormalities todetermine if leukemic clone still present on day 14.

Expected Outcomes

Patients receiving RARα selective agonist are monitored for responsecriteria based on hematological parameters including complete bloodcounts and percentage of leukemia blasts in the peripheral blood and inthe bone marrow. Patients with improved neutrophil count, decreasedtransfusion requirements of red blood cells and platelets together withdecreased percentage of blasts in the bone marrow and induction ofdifferentiation and apoptosis of these malignant blasts are deemedresponsive to therapy. Quality of life parameters such as pain,performance status and participation in activity of daily living andinstrumental activities of daily living are assessed to evaluate theimpact of this therapy on study patients. Use of this RARα selectiveagonist is expected to improve hematological and quality of lifeparameters in patients with AML and solid malignancies. In addition, theuse of the RARα agonist which is CYP26 resistant may result indifferentiation and thus elimination of minimal residual disease in thebone marrow of these patients.

Example 7 Clinical Study of IRX5183+CAR-Modified Immune Cells in AcuteMyeloid Leukemia

Eligibility criteria are the same as in Example 6.

Treatment Plan

The study uses the optimal dose of IRX5183 identified in thephase-escalation study (Example 6) and includes two separate arms; onefor patients receiving CAR-modified immune cells alone (Group 1) andanother for patient receiving both IRX5183 and CAR-modified immune cells(Group 2), and each of these two arms will recruit 26 patients.

All patients are administered fludarabine at 25 mg/m² intravenously ondays D-5 to D-3 and 900 mg/m² cyclophosphamide on day D-3.

Group 1 patients receive autologous CD33-CAR-T cells as disclosed andprepared in Minagawa et al. (PLoS One 12:e0172640, 2017) andadministered intravenously at a dose of 5×10⁶ cells/kg of patient bodyweight in a single bolus dose.

Group 2 patients are treated with daily IRX5183 for 30 days prior toinitiating autologous CD33-CAR-T cell therapy (D-30) and continued forsix months or until toxicity or progression occurs. CD33-CAR-T cells areadministered intravenously at a dose of 5×10⁶ cells/kg of patient bodyweight in a single bolus dose 30 days after initiation of IRX5183dosing.

Pharmacokinetics Analyses.

Plasma concentrations of IRX5183 are evaluated for the escalation andexpansion phases, targeting safe and effective retinoid levels bypharmacokinetics using LCM-MS (liquid chromatography-mass spectroscopytandem). Targeting peak levels of 1 μM should avoid systemic toxicity,while presumably preserving local BM niche retinoid levels. The plasmaconcentration of IRX5183 is obtained using a single 2 mL blood sample,pre-dose on day 14. Samples are shipped to, and analyzed by, thedesignated analytical laboratory.

Pharmacodynamics Analyses.

In addition to assessing standard clinical response criteria, BMcellular (normal HSCs and LSCs) concentrations, peripheral blood andbone marrow blast counts, markers of differentiation, apoptosis, andclonogenic growth are determined. A bone marrow aspirate and biopsy areobtained at baseline, on day 14, and at the end each of the first 4cycles of therapy. Differentiation is assessed using flow cytometry,comparing expression of differentiation markers on CD45 positive cellsand ALDHint LSCs on day 14 marrow versus baseline. FISH analysis is alsoconducted after each cycle for patients with baseline abnormalities todetermine if leukemic clone still present on day 14.

Expected Outcomes

Patients receiving RARα agonist+CD33-CAR-T therapy are monitored forresponse criteria based on hematological parameters including completeblood counts and percentage of leukemia blasts in the peripheral bloodand in the bone marrow. Patients with improved neutrophil count,decreased transfusion requirements of red blood cells and plateletstogether with decreased percentage of blasts in the bone marrow andinduction of differentiation and apoptosis of these malignant blasts aredeemed responsive to therapy. Quality of life parameters such as pain,performance status and participation in activity of daily living andinstrumental activities of daily living are assessed to evaluate theimpact of this therapy on study patients. Use of this RARαagonist+CD33-CAR-T therapy is expected to improve hematological andquality of life parameters in patients with AML. In addition, the use ofa RARα agonist which is CYP26 resistant in combination with CAR-Ttherapy may result in differentiation and thus elimination of minimalresidual disease in the bone marrow of these patients.

Example 8 Clinical Study of IRX5183+CAR-Modified Immune Cells inMultiple Myeloma

Multiple myeloma (MM) is a form of blood cancer that occurs when whiteblood cells known as plasma cells, which are typically found in the bonemarrow, grow out of control and develop into tumors. Approximately30,000 new cases of multiple myeloma will be diagnosed this year in theU.S. There are few known risk factors for developing this disease and itmay not cause signs or symptoms that would lead to a diagnosis until ithas advanced to the kidneys and other organs.

B-cell maturation antigen (BCMA) is expressed on all plasma cells,including cancerous plasma cells in MM. Autologous BCMA-CAR-T cells, andtheir use in MM are disclosed in Ali et al. (Ali S A et al. Blood128:1688-1700, 2016).

Inclusion Criteria:

-   -   Patients must have histologically confirmed MM by a pathologist,        with MM cells expressing BCMA, previously treated with 2+ prior        lines of therapy including an IMiD and a PI, either with        refractory, persistent, or progressive disease    -   Age ≥18 years of age    -   Creatinine ≤2.0 mg/dL, direct bilirubin ≤2.0 mg/dL, AST and ALT        ≤3.0× upper limit of normal (ULN)    -   Adequate pulmonary function as assessed by ≥92% oxygen        saturation on room air by pulse oximetry.

Exclusion Criteria:

-   -   Karnofsky performance status <70    -   Pregnant or lactating women. Women and men of childbearing age        should use effective contraception while on this study and        continue for 1 year after all treatment is finished    -   Impaired cardiac function (LVEF <40%) as assessed by ECHO or        MUGA scan    -   Patients with following cardiac conditions will be excluded:        -   New York Heart Association (NYHA) stage III or IV congestive            heart failure        -   Myocardial infarction months prior to enrollment        -   History of clinically significant ventricular arrhythmia or            unexplained syncope, not believed to be vasovagal in nature            or due to dehydration        -   History of severe non-ischemic cardiomyopathy    -   Patients with HIV or active hepatitis B or hepatitis C infection        are ineligible    -   Patients with any concurrent active malignancies as defined by        malignancies requiring any therapy other than expectant        observation or hormonal therapy, with the exception of squamous        and basal cell carcinoma of skin    -   Patients with a prior allogeneic transplant ARE eligible UNLESS        previously or currently experienced GvHD that required systemic        steroids or other systemic lymphotoxic therapy    -   Patients on systemic steroids (except if solely for adrenal        replacement) within two weeks of collection    -   Active autoimmune disease including connective tissue disease,        uveitis, sarcoidosis, inflammatory bowel disease, or multiple        sclerosis, or have a history of severe (as judged by the        principal investigator) autoimmune disease requiring prolonged        immunosuppressive therapy    -   Prior treatment with gene modified T cells    -   Prior or active CNS involvement by myeloma (eg leptomeningial        disease). Screening for this, for example, by lumbar puncture,        is only required if suspicious symptoms or radiographic findings        are present    -   Plasma cell leukemia    -   Pre-existing (active or severe) neurologic disorders (e.g.        pre-existing seizure disorder)    -   Active uncontrolled acute infections    -   Any other issue which, in the opinion of the treating physician,        would make the patient ineligible for the study

Modified T cell infusions are administered 2-7 days following thecompletion of conditioning chemotherapy (cyclophosphamide and optionallyfludarabine). BCMA-CAR-T cells are administered at a dose of 1-10×10⁶CAR-T cells/kg.

Conditioning chemotherapy comprises cyclophosphamide at 3000 mg/m² IVonce on day D-7 to D-2 or low intensity cy/flu (cyclophosphamide 300mg/m²/day×3+fludarabine 30 mg/m²/day×3) with the last day occurring onday D-7 to D-2.

The study uses two doses of IRX5183 in the range of 15-75 mg/m²/day(this range may be narrowed based on the results of the clinical trialin Example 6) and includes three separate arms; one for patientsreceiving BCMA-CAR-T cells alone (Group 1) and other for patientsreceiving both IRX5183 and BCMA-CAR-T cells at IRX5183 dose 1 (Group 2)and IRX5183 dose 2 (Group 3), and each of these two arms will recruit 26patients.

Group 1 patients are treated with BCMA-CAR-T intravenously at a dose of1-10×10⁶ cells/kg of patient body weight in a single bolus dosebeginning on DO after conditioning chemotherapy.

Group 2 and 3 patients are treated with daily IRX5183 for 30 days priorto initiating BCMA-CAR-T cells and continued for six months or untiltoxicity or progression occurs. The BCMA-CAR-T cells are administeredintravenously in a single bolus dose of 1-10×10⁶ cells/kg of patientbody weight.

Expected Outcomes

Patients receiving RARα agonist+BCMA-CAR-T therapy are monitored forresponse criteria based on duration presence of BCMA-CAR-T cells in thesubject, tumor growth, metastases, etc. Quality of life parameters suchas pain, performance status and participation in activity of dailyliving and instrumental activities of daily living are assessed toevaluate the impact of this therapy on study patients. Use of this RARαagonist+BCMA-CAR-T therapy is expected to improve quality of lifeparameters in patients with BCMA-expressing tumors and prevent diseaseprogression. In addition, the use of a RARα agonist which is CYP26resistant in combination with BCMA-CAR-T therapy may result indifferentiation and thus elimination of minimal residual disease in thebone marrow of these patients.

Example 9 Clinical Study of IRX5183+CAR-Modified Immune Cells inMesothelin-Expressing Solid Tumors

Mesothelin is a 40-KDa cell surface tumor differentiation antigen, whichderived from the 69-KDa precursor protein encoded by Mesothelin gene.The normal biological function of mesothelin almost remains unknown.Some studies suggest that mesothelin is the receptor of CA125/MUC16, andthe interaction between mesothelin-CA125 mediates cell adhesion and maybe a critical point in the metastatic of ovarian cancer. Mesothelinoverexpression promotes tumor cell proliferation and regional invasionand is associated with poor prognosis, such as worse recurrence-freesurvival and overall survival. As a tumor marker, soluble mesothelin inserum plays an important role in diagnosing and monitoring therapeuticeffect for patients with malignant pleural mesothelioma (MPM) andovarian cancer. Mesothelin is expressed at low levels in normal tissues,including pleura, pericardium, peritoneum, tunica vaginalis, but it isoverexpressed in various malignancies including MPM, ovarian cancers,pancreatic cancers, and non-small cell lung cancers. Due to the weakexpression in normal tissues and strong expression in several cancers,mesothelin is an attractive target for immune-based therapies.

Patients will receive a nonmyeloablative but lymphocyte-depletingpreparative regimen followed by IV infusion of autologousanti-mesothelin CAR engineered cells (meso-CAR-T cells; disclosed indisclosed in Adusumilli et al., Sci Transl. Med. 6(261):26ra151, 2014)plus low dose IV aldesleukin.

Peripheral blood monocuclear cells (PBMC) obtained by leukapheresis willbe cultured in order to stimulate T cell growth. Transduction isinitiated by exposure of approximately 1-5×10⁸ cells to retroviralsupernatant containing the anti-mesothelin CAR. Patients will receive anonmyeloablative but lymphocyte depleting regimen comprisingcyclophosphamide and fludarabine (25 mg/m²/day IVPB daily over 30 minfor 5 days and cyclophosphamide 60 mg/kg/day×2 days IV in 250 ml D5Wover 1 hr) followed by IV infusion of ex vivo CAR gene-transduced PBMCplus low dose aldesleukin (72,000 IU/kg IV over a period of 15 minapproximately every eight hours (+/−1 hour) beginning within 24 hours ofcell infusion and continuing for up to 5 days for a maximum of 15doses).

Eligibility

Patients who are 18 years of age or older must have metastatic orunresectable cancer that expresses mesothelin and the patient haspreviously received and have been a non-responder to, or recurred after,standard care. Patients may not have contraindications for low dosealdesleukin administration.

Inclusion Criteria:

-   -   Metastatic or unresectable measurable cancers that express        mesothelin. Epithelial mesotheliomas and pancreatic cancers do        not need to be assessed for mesothelin expression since all of        these tumors have been shown to express mesothelin. Other        metastatic or unresectable cancers must be shown to express        mesothelin as assessed by RT-PCR or immunochemistry on tumor        tissue. Bi-phasic mesotheliomas must express meothelin on        greater than 50% of the cells in the epithelial component.    -   Patients must have previously received at least one systemic        standard care (or effective salvage chemotherapy regimens) for        metastatic or unresectable disease, if known to be effective for        that disease, and have been either non-responders (progressive        disease) or have recurred.    -   Greater than or equal to 18 years of age and less than or equal        to 70 years of age.    -   Willing to sign a durable power of attorney.    -   Able to understand and sign the Informed Consent Document.    -   Clinical performance status of ECOG 0 or 1.    -   Patients of both genders must be willing to practice birth        control from the time of enrollment on this study and for up to        four months after treatment.    -   Serology:        -   Seronegative for HIV antibody.        -   Seronegative for hepatitis B antigen and seronegative for            hepatitis C antibody. If hepatitis C antibody test is            positive, then patient must be tested for the presence of            antigen by RT-PCR and be HCV RNA negative.    -   Women of child-bearing potential must have a negative pregnancy        test because of the potentially dangerous effects of the        treatment on the fetus.    -   Hematology:        -   Absolute neutrophil count greater than 1000/mm³ without the            support of filgrastim.        -   WBC (>3000/mm³).        -   Platelet count greater than 100,000/mm³.        -   Hemoglobin greater than 8.0 g/dl.    -   Chemistry:        -   Serum ALT/AST less than or equal to 2.5 times the upper            limit of normal.        -   Serum creatinine less than or equal to 1.6 mg/dl.        -   Total bilirubin less than or equal to 1.5 mg/dl, except in            patients with Gilbert's Syndrome who must have a total            bilirubin less than 3.0 mg/dl/    -   More than four weeks must have elapsed since any prior systemic        therapy at the time the patient receives the preparative        regimen, and patient's toxicities must have recovered to a grade        of 1 or less (except for toxicities such as alopecia or        vitilago).

Exclusion Criteria:

-   -   Patients with sarcomatoid mesothelioma    -   Women of child-bearing potential who are pregnant or        breastfeeding.    -   Patients with known brain metastases.    -   Patients receiving full-dose anti-coagulative therapy.    -   Active systemic infections (e.g., requiring anti-infective        treatment), coagulation disorders or any other major medical        illness.    -   Any form of primary immunodeficiency    -   Concurrent opportunistic infections.    -   Patients with diabetic retinopathy.    -   Concurrent systemic steroid therapy.    -   History of severe immediate hypersensitivity reaction to any of        the agents used in this study.    -   History of coronary revascularization or ischemic conditions.    -   Documented LVEF of less than or equal to 45% tested in patients        with:        -   Clinically significant atrial and/or ventricular arrhythmias            including but not limited to atrial fibrillation,            ventricular tachycardia, second or third degree hear block,            chest pain, or ischemic heart disease.        -   Age greater than or equal to 60 years old.    -   Documented FEV1 less than or equal to 50% predicted tested in        patients with:        -   A prolonged history of cigarette smoking (20 pk/year of            smoking within the past 2 years).        -   Symptoms of respiratory dysfunction.    -   Patients who are receiving any other investigational agents.

Conditioning chemotherapy comprises cyclophosphamide at 3000 mg/m² IVonce on day D-7 to D-2 or low intensity cy/flu (cyclophosphamide 300mg/m²/day×3+fludarabine 30 mg/m²/day×3) with the last day occurring onday D-7 to D-2.

The study uses two doses of IRX5183 in the range of 15-75 mg/m²/day(this range may be narrowed based on the results of the clinical trialin Example 6) and includes three separate arms; one for patientsreceiving meso-CAR-T cells alone (Group 1) and other for patientsreceiving both IRX5183 and meso-CAR-T cells at IRX5183 dose 1 (Group 2)and IRX5183 dose 2 (Group 3), and each of these two arms will recruit 26patients.

Group 1 patients are treated with meso-CAR-T intravenously at a dose of1-10×10⁶ cells/kg of patient body weight in a single bolus dosebeginning on DO after conditioning chemotherapy.

Group 2 and 3 patients are treated with daily IRX5183 for 30 days priorto initiating meso-CAR-T cells and continued for six months or untiltoxicity or progression occurs. The meso-CAR-T cells are administeredintravenously in a single bolus dose of 1-10×10⁶ cells/kg of patientbody weight.

Expected Outcomes

Patients receiving RARα agonist+meso-CAR-T therapy are monitored forresponse criteria based on duration presence of meso-CAR-T cells in thesubject, tumor growth, metastases, etc. Quality of life parameters suchas pain, performance status and participation in activity of dailyliving and instrumental activities of daily living are assessed toevaluate the impact of this therapy on study patients. Use of this RARαagonist+meso-CAR-T therapy is expected to improve quality of lifeparameters in patients with meso-expressing tumors and prevent diseaseprogression. In addition, the use of a RARα agonist which is CYP26resistant in combination with meso-CAR-T therapy may result indifferentiation and thus elimination of minimal residual disease in thebone marrow of these patients.

Example 10 Clinical Study of IRX5183+CAR-Modified Immune Cells+TargetedImmunotherapy in Lung Cancer

Inclusion criteria and experimental design will be substantially as inExample 9 as follows:

Group 1 meso-CAR-T Group 2 IRX5183 (optimal dose established in Example9) + meso-CAR-T Group 3 meso-CAR-T + PD-1 inhibition Group 4 IRX5193 +meso-CAR-T + PD-1 inhibition

Dosing, concomitant medications, and evaluation of outcome will be as inExample 9.

Example 11 Use of IRX5183 in the Manufacture of CAR-Modified T Cells

Autologous T lymphocytes are purified from the peripheral blood of thepatient and cultured with feeder cells (such as autologousantigen-presenting cells) and growth factors, such as IL-2.

During the activation process, the T cells are incubated with the viralvector encoding the CAR, and, after several days, the vector is washedout of the culture by dilution and/or medium exchange. The viral vectoruses viral machinery to attach to the patient cells, and, upon entryinto the cells, the vector introduces genetic material in the form ofRNA. In the case of CAR T cell therapy, this genetic material encodesthe CAR. The RNA is reverse-transcribed into DNA and permanentlyintegrates into the genome of the patient cells; therefore, CARexpression is maintained as the cells divide and are grown to largenumbers in the bioreactor. The CAR is then transcribed and translated bythe patient cells, and the CAR is expressed on the cell surface.Lentiviral vectors, which have a safer integration site profile thangammaretroviral vectors are commonly used in clinical trials of CAR Tcell therapies.

The transduced cells are then cultured to the desired numbers formultiple rounds of CAR-T therapy. The culture cells can be cryopreservedfor future use.

RARα agonists are included in the culture before and/or aftertransduction with the viral vector to facilitate growth and maintenanceof the CAR phenotypes

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used hereinthe terms “about” and “approximately” means within 10 to 15%, preferablywithin 5 to 10%. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth inthe specific examples are reported as precisely as possible. Anynumerical value, however, inherently contains certain errors necessarilyresulting from the standard deviation found in their respective testingmeasurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1-30. (canceled)
 31. A method of augmenting the production of chimericantigen receptor-modified immune cells (CAR-MIC) comprising culturingthe CAR-MIC in a culture medium comprising at least one differentiatingretinoic acid receptor (RAR) active agent, wherein the differentiatingRAR active agent is a RARα agonist, wherein the RARα agonist is a CYP26metabolism-resistant, RARα selective agonist having a structure ofgeneral formula (I)

wherein R¹ is H or C₁₋₆ alky, R² and R³ are independently H or F, and R⁴is a halogen.
 32. The method of claim 31, wherein the RARα agonist is acompound having a structure of formula (II):

wherein R¹ is H or C₁₋₆ alkyl.
 33. The method of claim 35, wherein theRARα agonist is a compound having a structure of formula (III):


34. The method of claim 31, wherein augmenting the production CAR-MICcomprises expansion of CAR-MIC numbers.
 35. The method of claim 31,wherein augmenting the production CAR-MIC comprises maintenance of theCAR phenotype.
 36. The method of claim 31, wherein the culture ofCAR-MIC is administered directly to a subject as CAR-MIC cancerimmunotherapy.
 37. The method of claim 31, wherein the culture ofCAR-MIC is cryopreserved for future administration to a subject asCAR-MIC cancer immunotherapy.
 38. The method of claim 31, wherein theCAR-MIC is a CAR-T cell.